Methods of processing lignocellulosic biomass using single-stage autohydrolysis pretreatment and enzymatic hydrolysis

ABSTRACT

Methods of processing lignocellulosic biomass to fermentable sugars are provided which rely on hydrothermal pretreatment. Soft lignocellulosic biomass feedstock is pretreated in a single-stage pressurized hydrothermal pretreatment to very low severity. The pre-treated biomass is hydrolysed, typically as a whole slurry, using enzymatic hydrolysis catalysed by an enzyme mixture comprising endoglucanase, exoglucanase, β-glucosidase, endoxylanase, and β-xylosidase activities at activity levels in nkat/g glucan of endoglucanase of at least 1100, exoglucanase of at least 280, β-glucosidase of at least 3000, endoxylanase of at least 1400, and β-xylosidase of at least 75, so as to produce a hydrolysate in which the yield of C5 monomers is at least 55% of the original xylose and arabinose content of the feedstock prior to pretreatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/DK2014/050030, filed Feb. 5, 2014, which International Applicationwas published by the International Bureau in English on Feb. 5, 2015,and claims priority from International Application No.PCT/DK2013/050256, filed Aug. 1, 2013, which applications are herebyincorporated in their entirety by reference in this application.

FIELD

The invention relates, in general, to methods of processinglignocellulosic biomass to fermentable sugars and, in particular, tomethods that rely on hydrothermal pretreatment.

BACKGROUND

Historical reliance on petroleum and other fossil fuels has beenassociated with dramatic and alarming increases in atmospheric levels ofgreenhouse gases. International efforts are underway to mitigategreenhouse gas accumulation, supported by formal policy directives inmany countries. One central focus of these mitigation efforts has beendevelopment of processes and technologies for utilization of renewableplant biomass to replace petroleum as a source of precursors for fuelsand other chemical products. The annual growth of plant-derived biomasson earth is estimated to approximate 1×10^11 metric tons per year dryweight. See Lieth and Whittaker (1975). Biomass utilization is, thus, anultimate goal in development of sustainable economy.

Fuel ethanol produced from sugar and starch based plant materials, suchas sugarcane, root and grain crops, is already in wide use, with globalproduction currently topping 73 billion liters per year. Ethanol hasalways been considered an acceptable alternative to fossil fuels, beingreadily usable as an additive in fuel blends or even directly as fuelfor personal automobiles. However, use of ethanol produced by these“first generation” bioethanol technologies does not actually achievedramatic reduction in greenhouse gas emission. The net savings is onlyabout 13% compared with petroleum, when the total fossil fuel inputs tothe final ethanol outputs are all accounted. See Farrell et al. (2006).Moreover, both economic and moral objections have been raised to the“first generation” bioethanol market. This effectively places demand forcrops as human food into direct competition with demand for personalautomobiles. And indeed, fuel ethanol demand has been associated withincreased grain prices that have proved troublesome for poor,grain-importing countries.

Great interest has arisen in developing biomass conversion systems thatdo not consume food crops—so-called “second generation” biorefining,whereby bioethanol and other products can be produced fromlignocellulosic biomass such as crop wastes (stalks, cobs, pits, stems,shells, husks, etc. . . . ), grasses, straws, wood chips, waste paperand the like. In “second generation” technology, fermentable 6-carbon(C6) sugars derived primarily from cellulose and fermentable 5-carbon(C5) sugars derived from hemicellulose are liberated from biomasspolysaccharide polymer chains by enzymatic hydrolysis or, in some cases,by pure chemical hydrolysis. The fermentable sugars obtained frombiomass conversion in a “second generation” biorefinery can be used toproduce fuel ethanol or, alternatively, other fuels such as butanol, orlactic acid monomers for use in synthesis of bioplastics, or many otherproducts.

The total yield of both C5 and C6 sugars is a central consideration incommercialization of lignocellulosic biomass processing. In the case ofethanol production, and also production of lactate or other chemicals,it can be advantageous to combine both C5 and C6 sugar process streamsinto one sugar solution. Modified fermentive organisms are now availablewhich can efficiently consume both C5 and C6 sugars in ethanolproduction. See e.g. Madhavan et al. (2012); Dumon et al. (2012); Hu etal. (2011); Kuhad et al. (2011); Ghosh et al. (2011); Kurian et al.(2010); Jojima et al. (2010); Sanchez et al. (2010); Bettiga et al.(2009); Matsushika et al. (2009).

Because of limitations of its physical structure, lignocellulosicbiomass cannot be effectively converted to fermentable sugars byenzymatic hydrolysis without some pretreatment process. A wide varietyof different pretreatment schemes have been reported, each offeringdifferent advantages and disadvantages. For review see Agbor et al.(2011); Girio et al. (2010); Alvira et al. (2010); Taherzadeh and Karimi(2008). From an environmental and “renewability” perspective,hydrothermal pretreatments are especially attractive. These utilizepressurized steam/liquid hot water at temperatures on the order of160-230° C. to gently melt hydrophobic lignin that is intricatelyassociated with cellulose strands, to solubilize a major component ofhemicellulose, rich in C5 sugars, and to disrupt cellulose strands so asto improve accessibility to productive enzyme binding. Hydrothermalpretreatments can be conveniently integrated with existing coal- andbiomass-fired electrical power generation plants to efficiently utilizeturbine steam and “excess” power production capacity.

In the case of hydrothermal processes, it is well known in the art, andhas been widely discussed, that pretreatment must be optimized betweenconflicting purposes. On the one hand, pretreatment should ideallypreserve hemicellulose sugar content, so as to maximize the ultimateyield of monomeric hemicellulose-derived sugars. Yet at the same time,pretreatment should sufficiently expose and pre-condition cellulosechains to susceptibility of enzymatic hydrolysis such that reasonableyields of monomeric cellulose-derived sugars can be obtained withminimal enzyme consumption. Enzyme consumption is also a centralconsideration in commercialization of biomass processing, which teeterson the verge of “economic profitability” in the context of “globalmarket economies” as these are currently defined. Notwithstandingdramatic improvements in recent years, the high cost of commerciallyavailable enzyme preparations remains one of the highest operating costsin biomass conversion.

As hydrothermal pretreatment temperatures and reactor residence timesare increased, a greater proportion of C5 sugars derived fromhemicellulose is irretrievably lost due to chemical transformation toother substances, including furfural and products of condensationreactions. Yet higher temperatures and residence times are required inorder to properly condition cellulose fibers for efficient enzymatichydrolysis to monomeric 6-carbon glucose.

In the prior art, an often used parameter of hydrothermal pretreatment“severity” is “R_(o),” which is typically referred to as a log value. Roreflects a composite measure of pretreatment temperature and reactorresidence time according to the equation: R_(o)=tEXP[T−100/14.75] wheret is residence time in minutes and T is reaction temperature in degreescentigrade.

Optimization of pretreatment conditions for any given biomass feedstockinherently requires some compromise between demands for high monomericC5 sugar yields from hemicellulose (low severity) and the demands forhigh monomeric C6 sugar yields from cellulose (high severity).

A variety of different hydrothermal pretreatment strategies have beenreported for maximizing sugar yields from both hemicellulose andcellulose and for minimizing xylo-oligomer inhibition of cellulasecatalysis. In some cases, exogenous acids or bases are added in order tocatalyse hemicellulose degradation (acid; pH<3.5) or ligninsolubilisation (base; pH>9.0). In other cases, hydrothermal pretreatmentis conducted using only very mild acetic acid derived fromlignocellulose itself (pH 3.5-9.0). Hydrothermal pretreatments underthese mild pH conditions have been termed “autohydrolysis” processes,because acetic acid liberated from hemicellulose esters itself furthercatalyses hemicellulose hydrolysis.

Acid catalysed hydrothermal pretreatments, known as “dilute acid” or“acid impregnation” treatments, typically provide high yields of C5sugars, since comparable hemicellulose solubilisation can occur at lowertemperatures in the presence of acid catalyst. Total C5 sugar yieldsafter dilute acid pretreatment followed by enzymatic hydrolysis aretypically on the order of 75% or more of what could theoretically beliberated from any given biomass feedstock. See e.g. Baboukaniu et al.(2012); Won et al. (2012); Lu et al. (2009); Jeong et al. (2010); Lee etal. (2008); Sassner et al. (2008); Thomsen et al. (2006); Chung et al.(2005).

Autohydrolysis hydrothermal pretreatments, in contrast, typicallyprovide much lower yields of C5 sugars, since higher temperaturepretreatment is required in the absence of acid catalyst. With theexception of autohydrolysis pretreatment conducted at commerciallyunrealistic low dry matter content, autohydrolysis treatments typicallyprovide C5 sugar yields <40% theoretical recovery. See e.g. Diaz et al.(2010); Dogaris et al. (2009). C5 yields from autohydrolysis as high as53% have been reported in cases where commercially unrealistic reactionstimes and extreme high enzyme doses were used. But even these very highC5 yields remain well beneath levels routinely obtained using diluteacid pretreatment. See e.g. Lee et al. (2009); Ohgren et al. (2007).

As a consequence of lower C5 yields obtained with autohydrolysis, mostreports concerning hydrothermal pretreatment in commercial biomassconversion systems have focused on dilute acid processes.Hemicellulose-derived C5 sugar yields on the order of 85% have beenachieved through use of so-called “two-stage” dilute acid pretreatments.In two-stage pretreatments, a lower initial temperature is used tosolubilize hemicellulose, whereafter the C5-rich liquid fraction isseparated. In the second stage, a higher temperature is used tocondition cellulose chains. See e.g. Mesa et al. (2011); Kim et al.(2011); Chen et al. (2010); Jin et al. (2010); Monavari et al. (2009);Soderstrom et al. (2005); Soderstrom et al. (2004); Soderstrom et al.(2003); Kim et al. (2001); Lee et al. (1997); Paptheofanous et al.(1995). One elaborate “two-stage” dilute acid pretreatment systemreported by the US National Renewable Energy Laboratory (NREL) claims tohave achieved C5 yields on the order of 90% using corn stover asfeedstock. See Humbird et al. (2011).

Notwithstanding the lower C5 yields which it provides, autohydrolysiscontinues to offer competitive advantages over dilute acid pretreatmentson commercial scale.

Most notable amongst the advantages of autohydrolysis processes is thatthe residual, unhydrolysed lignin has greatly enhanced market valuecompared with lignin recovered from dilute acid processes. First, thesulphuric acid typically used in dilute acid pretreatment imparts aresidual sulphur content. This renders the resulting lignin unattractiveto commercial power plants which would otherwise be inclined to consumesulphur-free lignin fuel pellets obtained from autohydrolysis as a“green” alternative to coal. Second, the sulfonation of lignin whichoccurs during sulphuric acid-catalysed hydrothermal pretreatmentsrenders it comparatively hydrophilic, thereby increasing its mechanicalwater holding capacity. This hydrophilicity both increases the cost ofdrying the lignin for commercial use and also renders it poorly suitedfor outdoor storage, given its propensity to absorb moisture. So-called“techno-economic models” of NREL's process for lignocellulosic biomassconversion, with dilute acid pretreatment, do not even account forlignin as a saleable commodity—only as an internal source of fuel forprocess steam. See Humbird et al. (2011). In contrast, the “economicprofitability” of process schemes that rely on autohydrolysis include asignificant contribution from robust sale of clean, dry lignin pellets.This is especially significant in that typical soft lignocellulosicbiomass feedstocks comprise a large proportion of lignin, between 10 and40% of dry matter content. Thus, even where process sugar yields fromautohydrolysis systems can be diminished relative to dilute acidsystems, overall “profitability” can remain equivalent or even better.

Autohydrolysis processes also avoid other well known disadvantages ofdilute acid. The requirement for sulphuric acid diverges from aphilosophical orientation favouring “green” processing, introduces asubstantial operating cost for the acid as process input, and creates aneed for elaborate waste water treatment systems and also for expensiveanti-corrosive equipment.

Autohydrolysis is also advantageously scalable to modest processingscenarios. The dilute acid process described by NREL is so complex andelaborate that it cannot realistically be established on a smallerscale—only on a gigantic scale on the order of 100 tons of biomassfeedstock per hour. Such a scale is only appropriate inhyper-centralized biomass processing scenarios. See Humbird et al.(2011). Hyper-centralized biomass processing of corn stover may well beappropriate in the USA, which has an abundance of genetically-engineeredcorn grown in chemically-enhanced hyper-production. But such a system isless relevant elsewhere in the world. Such a system is inappropriate formodest biomass processing scenarios, for example, on-site processing atsugar cane or palm oil or sorghum fields, or regional processing ofwheat straw, which typically produces much less biomass per hectare thancorn, even with genetic-engineering and chemical-enhancements.

Autohydrolysis systems, in contrast with dilute acid, are legitimately“green,” readily scalable, and unencumbered by requirements forelaborate waste water treatment systems. It is accordingly advantageousto provide improved autohydrolysis systems, even where these may not beobviously advantageous over dilute acid systems in terms of sugar yieldsalone.

The problem of poor C5 monomer yields with autohydrolysis has generallydriven commercial providers of lignocellulosic biomass processingtechnology to pursue other approaches. Some “two-stage” pretreatmentsystems, designed to provide improved C5 yields, have been reported withautohydrolysis pretreatments. See WO2010/113129; US2010/0279361; WO2009/108773; US2009/0308383; U.S. Pat. No. 8,057,639; US20130029406. Inthese “two stage” pretreatment schemes, some C5-rich liquid fraction isremoved by solid/liquid separation after a lower temperaturepretreatment, followed by a subsequent, higher temperature pretreatmentof the solid fraction. Most of these published patent applications didnot report actual experimental results. In its description of two-stageautohydrolytic pretreatment in WO2010/113129, Chemtex Italia reports atotal of 26 experimental examples using wheat straw with an average C5sugar recovery of 52%. These C5 recovery values do not distinguishbetween C5 recovery per se and monomer sugar yields, which is thesubstrate actually consumed in fermentation to ethanol and other usefulproducts.

The introduction of a second pretreatment stage into a scheme forprocessing lignocellulosic biomass introduces additional complexitiesand costs. It is accordingly advantageous to substantially achieve theadvantages of two-stage pretreatment using a simple single-stageautohydrolysis system.

We have discovered that, where single-stage autohydrolysis pretreatmentis conducted to very low severity, it is possible to achieveunexpectedly high final C5 monomer yields of 55% theoretical yield andhigher, while still achieving reasonable glucose yields. Where biomassfeedstocks are pretreated to such low severity that the undissolvedsolids content of pretreated material retains a residual xylan contentof at least 5.0% by weight, loss of C5 during pretreatment is minimized.Yet contrary to expectations, this very high residual xylan content canbe enzymatically hydrolysed to monomer xylose, with high recovery, whilesacrificing only a very small percentage of cellulose conversion toglucose, provided that sufficiently high xylanase and xylosidaseactivities are employed during enzymatic hydrolysis.

At these very low severity levels, the production of soluble by-productsthat affect cellulase activity or fermentive organisms is kept so lowthat the pretreated material can be used directly in enzymatichydrolysis, and subsequent fermentation, typically without requirementfor any washing or other de-toxification step.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows xylan number as a function of pretreatment severity factorfor soft lignocellulosic biomass feedstocks subject to autohydrolysispretreatment.

FIG. 2 shows the percentage by weight xylan in undissolved solids as afunction of xylan number for pretreated feedstocks.

FIG. 3 shows C5 recovery in soluble and insoluble form as a function ofxylan number for soft lignocellulosic biomass feedstocks subject toautohydrolysis pretreatment.

FIG. 4 shows total C5 recovery as a function of xylan number for softlignocellulosic biomass feedstocks subject to autohydrolysispretreatment.

FIG. 5 shows production of acetic acid, furfural and 5-HMF as a functionof xylan number for soft lignocellulosic biomass feedstocks subject toautohydrolysis pretreatment.

FIG. 6 shows the effect of removal of dissolved solids on celluloseconversion for soft lignocellulosic biomass feedstocks subject to verylow severity autohydrolysis pretreatment.

FIG. 7 shows HPLC characterization of liquid fraction from softlignocellulosic biomass feedstocks subject to very low severityautohydrolysis pretreatment.

FIG. 8 shows C5 sugar recovery as a function of time where solidfraction is subject to enzymatic hydrolysis followed by introduction ofliquid fraction for post-hydrolysis.

FIG. 9 shows fermentation profile of ethanol fermentation by a modifiedyeast strain using wheat straw that was pretreated by very low severityautohydrolysis, enzymatically hydrolysed and used as combined liquid andsolid fraction without de-toxification to remove fermentationinhibitors.

FIG. 10 shows a process scheme for one embodiment.

FIG. 11 show cellulose conversion as a function of time—C5 bypass.

FIG. 12 shows Xylan conversion as a function of time—C5 bypass.

FIG. 13 show cellulose conversion as a function of time—whole slurry.

FIG. 14 shows xylan conversion as a function of time—whole slurry.

FIG. 15 shows total C6 and C5 recovery after pretreatment and hydrolysisas a function of hydrolysis time—whole slurry.

DETAILED DESCRIPTION OF EMBODIMENTS

In some embodiments the invention provides methods of processinglignocellulosic biomass comprising:

-   -   Providing soft lignocellulosic biomass feedstock,    -   Pretreating the feedstock at pH within the range 3.5 to 9.0 in a        single-stage pressurized hydrothermal pretreatment to log        severity Ro 3.75 or lower so as to produce a pretreated biomass        slurry in which the undissolved solids comprise at least 5.0% by        weight xylan, and    -   Hydrolysing the pre-treated biomass with or without addition of        supplemental water content using enzymatic hydrolysis for at        least 24 hours catalysed by an enzyme mixture comprising        endoglucanase, exoglucanase, β-glucosidase, endoxylanase, and        β-xylosidase activities at activity levels in nkat/g glucan of        endoglucanase of at least 1100, exoglucanase of at least 280,        β-glucosidase of at least 3000, endoxylanase of at least 1400,        and β-xylosidase of at least 75, so as to produce a hydrolysate        in which the yield of C5 monomers is at least 55% of the        original xylose and arabinose content of the feedstock prior to        pretreatment.

In some embodiments, the pretreated biomass is hydrolysed as a wholeslurry comprising both solid and liquid fractions.

As used herein, the following terms have the following meanings:

“About” as used herein with reference to a quantitative number or rangerefers to +/−10% in relative terms of the number or range referred to.

“Autohydrolysis” refers to a pretreatment process in which acetic acidliberated by hemicellulose hydrolysis during pretreatment furthercatalyzes hemicellulose hydrolysis, and applies to any hydrothermalpretreatment of lignocellulosic biomass conducted at pH between 3.5 and9.0.

“Commercially available cellulase preparation optimized forlignocellulosic biomass conversion” refers to a commercially availablemixture of enzyme activities that is sufficient to provide enzymatichydrolysis of pretreated lignocellulosic biomass and that comprisesendocellulase (endoglucanase), exocellulase (exoglucanase),endoxylanase, xylosidase and B-glucosidase activities. The term“optimized for lignocellulosic biomass conversion” refers to a productdevelopment process in which enzyme mixtures have been selected and/ormodified for the specific purpose of improving hydrolysis yields and/orreducing enzyme consumption in hydrolysis of pretreated lignocellulosicbiomass to fermentable sugars.

Conducting pretreatment “at” a dry matter level refers to the dry mattercontent of the feedstock at the start of pressurized hydrothermalpretreatment. Pretreatment is conducted “at” a pH where the pH of theaqueous content of the biomass is that pH at the start of pressurizedhydrothermal pretreatment.

“Dry matter,” also appearing as DM, refers to total solids, both solubleand insoluble, and effectively means “non-water content.” Dry mattercontent is measured by drying at 105° C. until constant weight isachieved.

“Fiber structure” is maintained to the extent that the average size offiber fragments following pretreatment is >750 um.

“Glucan” as used herein refers to cellulose as well as othergluco-oligomers.

“Hydrothermal pretreatment” refers to the use of water, either as hotliquid, vapor steam or pressurized steam comprising high temperatureliquid or steam or both, to “cook” biomass, at temperatures of 120° C.or higher, either with or without addition of acids or other chemicals.

“Single-stage pressurized hydrothermal pretreatment” refers to apretreatment in which biomass is subject to pressurized hydrothermalpretreatment in a single reactor configured to heat biomass in a singlepass and in which no further pressurized hydrothermal pretreatment isapplied following a solid/liquid separation step to remove liquidfraction from feedstock subject to pressurized hydrothermalpretreatment.

“Solid/liquid separation” refers to an active mechanical process wherebyliquid is separated from solid by application of force through pressing,centrifugal or other force.

“Soft lignocellulosic biomass” refers to plant biomass other than woodcomprising cellulose, hemicellulose and lignin.

“Solid fraction” and “Liquid fraction” refer to fractionation ofpretreated biomass in solid/liquid separation. The separated liquid iscollectively referred to as “liquid fraction.” The residual fractioncomprising considerable insoluble solid content is referred to as “solidfraction.” A “solid fraction” will have a dry matter content andtypically will also comprise a considerable residual of “liquidfraction.”

“Theoretical yield” refers to the molar equivalent mass of pure monomersugars obtained from polymeric cellulose, or from polymerichemicellulose structures, in which constituent monomeric sugars may alsobe esterified or otherwise substituted. “C5 monomer yields” as apercentage of theoretical yield are determined as follows: Prior topretreatment, biomass feedstock is analysed for carbohydrates using thestrong acid hydrolysis method of Sluiter et al. (2008) using an HPLCcolumn and elution system in which galactose and mannose co-elute withxylose. Examples of such systems include a REZEX™ Monossacharide H+column from Phenomenex and an AMINEX HPX 87C™ column from Biorad. Duringstrong acid hydrolysis, esters and acid-labile substitutions areremoved. Except as otherwise specified, the total quantity of“Xylose”+Arabinose determined in the un-pretreated biomass is taken as100% theoretical C5 monomer recovery, which can be termed collectively“C5 monomer recovery.” Monomer sugar determinations are made using HPLCcharacterization based on standard curves with purified externalstandards. Actual C5 monomer recovery is determined by HPLCcharacterization of samples for direct measurement of C5 monomers, whichare then expressed as a percent of theoretical yield.

“Xylan number” refers to a characterization of pretreated biomassdetermined as follows: Pretreated biomass is obtained at about 30% totalsolids, typically after a solid/liquid separation to provide a solidfraction and a liquid fraction. This pretreated biomass having about 30%total solids is then partially washed by mixing with 70° C. water in theratio of total solids (DM) to water of 1:3 wt:wt. The pretreated biomasswashed in this manner is then pressed to about 30% total solids. Xylancontent of the pretreated biomass washed in this manner and pressed toabout 30% total solids is determined using the method of A. Sluiter, etal., “Determination of structural carbohydrates and lignin in biomass,”US National Renewable Energy Laboratory (NREL) Laboratory AnalyticalProcedure (LAP) with issue date Apr. 25, 2008, as described in TechnicalReport NREL/TP-510-42618, revised April 2008, which is expresslyincorporated by reference herein in entirety. An HPLC column and elutionsystem is used in which galactose and mannose co-elute with xylose.Examples of such systems include a REZEX™ Monossacharide H+ column fromPhenomenex and an AMINEX HPX 87C™ column from Biorad. This measurementof xylan content as described will include some contribution of solublematerial from residual liquid fraction that is not washed out of solidfraction under these conditions. Accordingly, “xylan number” provides a“weighted combination” measurement of residual xylan content withininsoluble solids and of soluble xylose and xylo-oligomer content withinthe “liquid fraction.”

The xylan content of undissolved solids is determined by taking arepresentative sample of pretreated biomass, subjecting it tosolid/liquid separation so as to provide a solid fraction having atleast 30% total solids. This pretreated biomass having at least 30%total solids is then partially washed by mixing with 70° C. water in theratio of total solids (DM) to water of 1:3 wt:wt. The pretreated biomasswashed in this manner is then pressed to at least 30% total solids andagain washed by mixing with 70° C. water in the ratio of total solids(DM) to water of 1:3 wt:wt. This washed material is then again pressedto at least 30% total solids and again washed by mixing with 70° C.water in the ratio of total solids (DM) to water of 1:3 wt:wt. Thiswashed material is then again pressed to at least 30% total solids andused as the sample of undissolved solids to be analysed. Xylan contentof the undissolved solids sample is then determined as described in theexplanation above concerning determination of xylan number and expressedas a percentage by weight of the total solids content of the sample.

Any suitable soft lignocellulosic biomass may be used, includingbiomasses such as at least wheat straw, corn stover, corn cobs, emptyfruit bunches, rice straw, oat straw, barley straw, canola straw, ryestraw, sorghum, sweet sorghum, soybean stover, switch grass, Bermudagrass and other grasses, bagasse, beet pulp, corn fiber, or anycombinations thereof. Lignocellulosic biomass may comprise otherlignocellulosic materials such as paper, newsprint, cardboard, or othermunicipal or office wastes. Lignocellulosic biomass may be used as amixture of materials originating from different feedstocks, may befresh, partially dried, fully dried or any combination thereof. In someembodiments, methods of the invention are practiced using at least about10 kg biomass feedstock, or at least 100 kg, or at least 500 kg.

Lignocellulosic biomass comprises crystalline cellulose fibrilsintercalated within a loosely organized matrix of hemicellulose andsealed within an environment rich in hydrophobic lignin. While celluloseitself comprises long, straight chain polymers of D-glucose,hemicellulose is a heterogeneous mixture of short, branched-chaincarbohydrates including monomers of all the 5-carbon aldopentoses (C5sugars) as well as some 6-carbon (C6) sugars including glucose andmannose. Lignin is a highly heterogeneous polymer, lacking anyparticular primary structure, and comprising hydrophobic phenylpropanoidmonomers.

Suitable lignocellulosic biomass typically comprises cellulose inamounts between 20 and 50% of dry mass prior to pretreatment, lignin inamounts between 10 and 40% of dry mass prior to pretreatment, andhemicellulose in amounts between 15 and 40%.

In some embodiments, biomass feedstocks may be subject to particle sizereduction and/or other mechanical processing such as grinding, milling,shredding, cutting or other processes prior to hydrothermalpretreatment. In some embodiments, biomass feedstocks may be washedand/or leached of valuable salts prior to pressurized pretreatment, asdescribed in Knudsen et al. (1998). In some embodiments feedstocks maybe soaked prior to pressurized pretreatment at temperatures up to 99° C.

In some embodiments the feedstock is first soaked in an aqueous solutionprior to hydrothermal pretreatment. In some embodiments, it can beadvantageous to soak the feedstock in an acetic acid containing liquidobtained from a subsequent step in the pretreatments, as described inU.S. Pat. No. 8,123,864, which is hereby incorporated by reference inentirety. It is advantageous to conduct treatment at the highestpossible dry matter content, as described in U.S. Ser. No. 12/935,587,which is hereby incorporated by reference in entirety. Conductingpretreatment at high dry matter avoids expenditure of process energy onheating of unnecessary water. However, some water content is required toachieve optimal eventual sugar yields from enzymatic hydrolysis.Typically it is advantageous to pretreat biomass feedstocks at or closeto their inherent water holding capacity. This is the level of watercontent that a given feedstock will attain after soaking in an excess ofwater followed by pressing to the mechanical limits of an ordinarycommercial screw press—typically between 30 and 45% DM. In someembodiments, hydrothermal pretreatment is conducted at DM content atleast 35%. It will be readily understood by one skilled in the art thatDM content may decrease during hydrothermal pretreatment as some watercontent is added during heating. In some embodiments, feedstocks arepretreated at DM content at least 20%, or at least 25%, or at least 30%,or at least 40%, or 40% or less, or 35% or less, or 30% or less.

In some embodiments, soaking/wetting with an aqueous solution can serveto adjust pH prior to pretreatment to the range of between 3.5 and 9.0,which is typically advantageous for autohydrolysis. It will be readilyunderstood that pH may change during pretreatment, typically to moreacidic levels as acetic acid is liberated from solubilizedhemicellulose.

In some embodiments, hydrothermal pretreatment is conducted withoutsupplemental oxygen as required for wet oxidation pretreatments, orwithout addition of organic solvent as required for organosolvpretreatment, or without use of microwave heating as required formicrowave pretreatments. In some embodiments, hydrothermal pretreatmentis conducted at temperatures of 140° C. or higher, or at 150° C. orhigher, or at 160° C. or higher, or between 160 and 200° C., or between170 and 190° C., or at 180° C. or lower, or at 170° C. or lower.

In some embodiments, some C5 content may be removed by a soaking stepprior to pressurized pretreatment. In some embodiments, the singlereactor may be configured to heat biomass to a single targettemperature. Alternatively, the single reactor may be configured toaffect a temperature gradient within the reactor such that biomass isexposed, during a single passage, to more than one temperature region.In some embodiments, it may be advantageous to partially remove somesolubilized biomass components from within the pressurized reactorduring the course of pretreatment.

Suitable hydrothermal pretreatment reactors typically include mostpulping reactors known from the pulp and paper industry. In someembodiments, hydrothermal pretreatment is administered by steam within areactor pressurized to 10 bar or lower, or to 12 bar or lower, or to 4bar or higher, or 8 bar or higher, or between 8 and 18 bar, or between18 and 20 bar. In some embodiments, the pretreatment reactor isconfigured for a continuous inflow of feedstock.

In some embodiments, wetted biomass is conveyed through the reactor,under pressure, for a certain duration or “residence time.” Residencetime is advantageously kept brief to facilitate higher biomassthroughput. However, the pretreatment severity obtained is determinedboth by temperature and also by residence time. Temperature duringhydrothermal pretreatment is advantageously kept lower, not only becausemethods of the invention seek to obtain a very low pretreatmentseverity, but also because lower temperatures can be accomplished usinglower steam pressures. To the extent that pretreatment temperature canbe at levels of 180° C. or lower, and accordingly, saturated steampressures kept to 10 bar or lower, lower tendency for corrosion isexperienced and much lower grade pressure fittings and steelcompositions may be used, which reduces plant capital costs. In someembodiments, the reactor is configured to heat biomass to a singletarget temperature between 160 and 200° C., or between 170 and 190° C.Residence times in some embodiments are less than 60, or less than 30,or less than 20, or less than 15, or less than 14, or less than 13, orless than 12, or less than 10, or less than 8, or less than 5 minutes.

Biomass feedstocks may be loaded from atmospheric pressure into apressurized reactor by a variety of means. In some embodiments, asluice-type “particle pump” system may be used to load biomassfeedstocks, such as the system described in U.S. Ser. No. 13/062,522,which is hereby incorporated by reference in entirety. In someembodiments, it may be advantageous to load a pretreatment reactor usinga so-called “screw plug” feeder.

Pretreated biomass may be unloaded from a pressurized reactor by avariety of means. In some embodiments, pretreated biomass is unloaded insuch manner as to preserve the fiber structure of the material.Preserving the fiber structure of the pretreated biomass is advantageousbecause this permits the solid fraction of the pretreated material to bepressed during solid/liquid separation to comparatively high dry matterlevels using ordinary screw press equipment, and thereby avoiding theadded expense and complexity of membrane filter press systems.

Fiber structure can be maintained by removing the feedstock from thepressurized reactor in a manner that is non-explosive. In someembodiments, non-explosive removal may be accomplished and fiberstructure thereby maintained using a sluice-type system, such as thatdescribed in U.S. Ser. No. 13/043,486, which is hereby incorporated byreference in entirety. In some embodiments, non-explosive removal may beaccomplished and fiber structure thereby maintained using a hydrocycloneremoval system, such as those described in U.S. Ser. No. 12/996,392,which are hereby incorporated by reference in entirety.

In some embodiments, pretreated biomass can be removed from apressurized pretreatment reactor using “steam explosion,” which involvesexplosive release of the pretreated material. Steam-exploded, pretreatedbiomass does not retain its fiber structure and accordingly requiresmore elaborate solid/liquid separation systems in order to achieve drymatter content comparable to that which can be achieved using ordinaryscrew press systems with pretreated biomass that retains its fiberstructure.

The biomass feedstock is pretreated to very low severity log Ro 3.75 orless. This will typically result in the pretreated biomass having axylan number of 10% or higher. The parameter “xylan number” refers to acomposite measurement that reflects a weighted combination of bothresidual xylan content remaining within insoluble solids and also theconcentration of soluble xylose and xylo-oligomers within the liquidfraction. At lower Ro severity, xylan number is higher. Thus, thehighest xylan number refers to the lowest pretreatment severity. Xylannumber provides a negative linear correlation with the conventionalseverity measure log R_(o) even to very low severity, where residualxylan content within undissolved solids is higher. The relationshipbetween pretreatment severity log Ro and xylan number of the resultingpretreated biomass for a variety of different feedstocks is shown inFIG. 1, which is explained in detail in Example 1.

Xylan number is particularly useful as a measure of pretreatmentseverity in that different pretreated biomass feedstocks havingequivalent xylan number exhibit equivalent C5 monomer recovery. Incontrast, conventional Ro severity is simply an empirical description ofpretreatment conditions, which does not provide a rational basis forcomparisons between different biomass feedstocks. For example, as shownin FIG. 1, single-stage autohydrolysis to severity log R_(o)=3.75provides pretreated sugar cane bagasse and corn stover having a xylannumber of between 6-7%, while with typical wheat straw strains, theresulting xylan number of pretreated feedstock is about 10%.

One skilled in the art can readily determine an appropriate pretreatmentseverity log Ro for any given feedstock to produce a pretreated biomasshaving the desired xylan number. In some embodiments biomass feedstockis pretreated to very low severity log Ro 3.75 or less, or 3.74 or less,or 3.73 or less, or 3.72 or less, or 3.71 or less, or 3.70 or less, or3.69 or less, or 3.68 or less, or 3.67 or less, or 3.66 or less, or 3.65or less, or 3.64 or less, or 3.63 or less, or 3.62 or less, or 3.61 orless, or 3.60 or less, or 3.59 or less, or 3.58 or less, or 3.57 orless, or 3.56 or less, or 3.55 or less, or 3.54 or less, or 3.53 orless, or 3.52 or less, or 3.51 or less, or 3.50 or less, or 3.45 orless, or 3.40 or less. In some embodiments, the biomass is pretreated toa low severity log Ro so as to produce a pretreated biomass having axylan number of 11% or higher, or 12% or higher, or 13% or higher, or14% or higher, or 15% or higher, or 16% or higher, or 17% or higher.

Xylan number is useful as a practical measure in production scalebiomass processing because it can be readily measured on-line based onsimple measurements such as are provided by near infrared spectroscopicmonitors. Xylan number can further be used as an end-point measure forcontrol of pretreatment, as described in WO2013/120492.

As an alternative to xylan number, which reflects a composite measure ofboth soluble xylan content and also residual xylan content inundissolved solids, the effect of pretreatment can be expressed in termsof residual xylan content in undissolved solids. The relationshipbetween residual xylan content in undissolved solids and xylan number isshown in FIG. 2, which is explained in Example 1. One skilled in the artcan readily determine an appropriate pretreatment severity log Ro forany given feedstock to produce a pretreated biomass having the desiredresidual xylan content in undissolved solids. For example, as shown inFIG. 2, single-stage autohydrolysis at a severity sufficient to producepretreated biomass having a xylan number of 10% will typically producepretreated biomass having a residual xylan content in undissolved solidsof about 5.0% by weight for soft lignocellulosic feedstocks, includingbut not limited to wheat straw, sugar cane bagasse, empty fruit bunches,and corn stover. In some embodiments, the biomass feedstock ispretreated to severity log Ro appropriate to produce a pretreatedbiomass in which the residual xylan content of undissolved solids is atleast 5.0% by weight, or at least 5.1%, or at least 5.2%, or at least5.3%, or at least 5.4%, or at least 5.5%, or at least 5.6%, or at least5.7%, or at least 5.8%, or at least 5.9%, or at least 6.0%, or at least6.1%, or at least 6.2%, or at least 6.3%, or at least 6.4%, or at least6.5%, or at least 6.6%, or at least 6.7%, or at least 6.8%, or at least6.9%, or at least 7.0%, or at least 7.1%, or at least 7.2%, or at least7.3% or at least 7.4%, or at least 7.5%, or at least 7.6%, or at least7.7%, or at least 7.8%, or at least 7.9% or at least 8.0%, or at least8.1%, or at least 8.2%, or at least 8.3%, or at least 8.4%, or at least8.5%.

It is advantageous that biomass feedstocks be pretreated to very lowseverity wherein xylan number of the pretreated feedstock is 10% orgreater, or wherein the residual xylan content of undissolved solids inthe pretreated feedstock is at least 5.0% or greater. This very lowseverity level corresponds to a process in which the total hemicellulosecontent of the feedstock before pretreatment that is either solubilizedor irretrievably lost during pretreatment is minimized. We haveunexpectedly discovered that very high final C5 monomer yields of atleast 55% theoretical can be obtained without appreciable loss of C6monomer yields with typical strains of wheat straw, sugar cane bagasse,sweet sorghum bagasse, corn stover, and empty fruit bunches afterenzymatic hydrolysis of feedstocks pretreated to very low severity bysingle-stage autohydrolysis, provided that sufficiently high xylanaseand xylosidase activities are employed during enzymatic hydrolysis. Atvery low severity levels, a large fraction of the feedstock'shemicellulose content remains within the solid fraction afterpretreatment, where it can subsequently be hydrolysed to C5 monomerswith high recovery using enzymatic hydrolysis, where sufficient xylanaseand xylosidase activities are used.

It should be noted that reports concerning “xylose recovery” are oftenexpressed in terms that are not comparable to the xylose recoveriesreported here. For example, Ohgren et al. (2007) and Lee et al. (2009)report high xylose recoveries. But these values refer only to xyloserecovery from pretreated biomass, not expressed as a percentage of theoriginal hemicellulose content of the feedstock prior to pretreatment.Or for example WO2010/113129 refers to hemicellulose recovery as apercentage of hemicellulose content of the feedstock prior topretreatment, but does not specify the monomer yield, which isinvariable smaller than the total hemicellulose recovery. In someembodiments, C5 monomer yields of at least 56% theoretical can beobtained in hydrolysate after enzymatic hydrolysis, or at least 57%, orat least 58%, or least 59%, or at least 60%, or at least 61%, or atleast 62%, or at least 63%, or at least 64%, or at least 65%.

Enzymatic hydrolysis may be conducted in a variety of different ways. Insome embodiments, the pretreated biomass is hydrolysed as a wholeslurry, meaning that substantially all of the solids from the pretreatedbiomass are subject to enzymatic hydrolysis in one reaction mixturecomprising both dissolved and undissolved solids. As used herein theterm “whole slurry” refers to an enzymatic hydrolysis reaction mixturein which the ratio by weight of undissolved to dissolved solids at thestart of enzymatic hydrolysis is less than 2.2:1.

Undissolved solids” and “dissolved solids” content of pretreated biomassslurry is determined as follows:

The “total solids” and “filtrated total solids” content are determinedaccording to the procedure described in Weiss et al. (2009). From thosevalues the “undissolved solids” and “dissolved solids” content can becalculated according to the following formulas:[Undissolved solids](wt-%)=([Total solids](wt-%)−[Filtrated totalsolids](wt-%))/(1−[Filtrated total solids](wt-%))[Dissolved solids](wt-%)=[Total solids](wt-%)−[Undissolved solids](wt-%)

In some embodiments, prior to enzymatic hydrolysis, the pretreatedbiomass is subject to a solid/liquid separation step to produce aseparate solid fraction and liquid fraction. Such a separation isgenerally advantageous in that some component of the dissolved solids inthe pretreated biomass typically acts to inhibit activities of one ormore enzymes used in enzymatic hydrolysis. For example, removal ofdissolved solids content from the pretreated biomass whole slurryclearly improves cellulose conversion achieved with feedstockspretreated as described here that were subject to enzymatic hydrolysisat high dry matter content using commercially available cellulasepreparations optimized for lignocellulosic biomass conversion providedeither by GENENCOR™ under the trademark ACCELLERASE TRIO™ or byNOVOZYMES™ under the trademark CELLIC CTEC3™, as is shown in FIG. 6 andas explained in Example 4.

Surprisingly, however, where the pretreated biomass slurry issufficiently diluted to lower dry matter content, the concentrations ofthe inhibiting substances present in the pretreated biomass slurry aresufficiently diluted that equivalent conversion yields can be obtainedin whole slurry hydrolysis, and even at lower enzyme dose levels, asexplained in Example 10.

In embodiments which employ a solid/liquid separation step prior toenzymatic hydrolysis, in which enzymatic hydrolysis is desired to beconducted at high dry matter content, it is advantageous to achieve thehighest practicable levels of dry matter content in the solid fractionor, alternatively, to remove the highest practicable amount of dissolvedsolids from the solid fraction. In some embodiments, solid/liquidseparation achieves a solid fraction having a DM content of at least 40%by weight, or at least 45%, or at least 50%, or at least 55%.Solid/liquid separation using ordinary screw press systems can typicallyachieve DM levels as high as 50% in the solid fraction, provided thebiomass feedstock has been pretreated in such manner that fiberstructure is maintained. In some embodiments, it may be advantageous toincur higher plant capital expenses in order to achieve more effectivesolid/liquid separation, for example, using a membrane filter presssystem. In some embodiments, dissolved solids can be removed from asolid fraction by serial washing and pressing or by displacement washingtechniques known in the pulp and paper art. In some embodiments, eitherby solid/liquid separation directly, or by some combination of washingand solid/liquid separation, the dissolved solids content of the solidfraction is reduced by at least 50%, or at least 55%, or at least 60%,or at least 65%, or at least 70%, or at least 75%.

The liquid fraction obtained from such a solid/liquid separation canthen be maintained separately from solid fraction during enzymatichydrolysis of the solid fraction. We term this temporary separation “C5bypass.” The C5-rich “bypass” material can then be added back into thehydrolysis mixture, after enzymatic hydrolysis of the solid fraction hasreached a desired degree of glucan conversion. We refer to this as“poshydrolysis” of the “C5 bypass” material. In this manner,interference that would otherwise be caused by the enzyme-inhibitorydissolved solids present in the pretreated biomass slurry is minimized,without loss of C5 monomer yield. Liquid fraction obtained from softlignocellulosic biomass feedstocks such as typical strains of wheatstraw, sugar cane bagasse, sweet sorghum bagasse, corn stover, and emptyfruit bunches pretreated by single-stage autohydrolysis to very lowseverity so as to provide pretreated material having xylan number 10% orhigher, or having undissolved solids xylan content of 5.0% or greater,typically comprise a small component of C6 monomers (1×), primarilyglucose with some other sugars; a larger component of soluble C6oligomers (about 2×-7×); a larger component of C5 monomers (about4×-8×), primarily xylose with some arabinose and other sugars; and amuch larger component of soluble xylo-oligomers (about 18×-30×). Solublexylo-oligomers typically include primarily xylohexose, xylopentose,xylotetraose, xylotriose and xylobiose with some higher chain oligomers.These xylose oligomers (or xylo-oligomers) are typically enzymaticallyhydrolysed during “posthydrolysis” by enzyme activities used in theenzymatic hydrolysis of solid fraction. Or in other words, by mixing theseparated liquid fraction and the hydrolysed solid fraction,xylo-oligomers in the liquid fraction are degraded to xylose monomers bythe action of enzyme activities remaining within the hydrolysed solidfraction.

Alternatively, in some embodiments, the separated liquid fraction can beused for other purposes. In some embodiments, the separated liquidfraction can be blended with thin stillage obtained after recovery ofethanol from fermentation of hydrolysates. The blended liquid fractionand thin stillage can then be used as a biomethane substrate. Oralternatively, the thin stillage and liquid fractions can be used asseparate biomethane substrate. Surprisingly, biomethane potential ofthin stillage and also of liquid fraction is increased after very lowseverity pretreatment that produce pretreated biomass having xylannumber 10% or greater. Consequently in some embodiments it isadvantageous to conduct C6 fermentation at very low severity, in thatincreased biomethane yields can offset somewhat decreased ethanolyields. In some embodiments, a biomethane yield of at least 75 NM3methane/ton biomass feedstock, or at least 78, or at least 80, or atleast 82, or at least 85, or at least 87, or at least 90, or at least92, or at least 95 can be produced from a biomethane substratecomprising combined liquid fraction and thin stillage, where feedstockhas been pretreated to low severity log Ro so as to produce a pretreatedbiomass having xylan number at least 10%, or in which the undissolvedsolids comprise at least 5.0% by weight xylan. In some embodiments, thewhole slurry hydrolysate itself is used as a biomethane substrate.

In some embodiments the invention provides methods of processinglignocellulosic biomass comprising:

-   -   Providing soft lignocellulosic biomass feedstock,    -   Pretreating the feedstock at pH within the range 3.5 to 9.0 in a        single-stage pressurized hydrothermal pretreatment to very low        severity such that the pretreated biomass is characterized by        having a xylan number of 10% or higher,    -   Separating the pretreated biomass into a solid fraction and a        liquid fraction,    -   Hydrolysing the solid fraction with or without addition of        supplemental water content using enzymatic hydrolysis catalysed        by an enzyme mixture comprising endoglucanase, exoglucanase,        B-glucosidase, endoxylanase, and xylosidase activities, and    -   Subsequently mixing the separated liquid fraction and the        hydrolysed solid fraction, whereby xylo-oligomers in the liquid        fraction are degraded to xylose monomers by the action of enzyme        activities remaining within the hydrolysed solid fraction.

In some embodiments, the enzymatic hydrolysate, either from a separatedsolid fraction or from a hydrolysed solid fraction to which separatedliquid fraction has been added for posthydrolysis, or from a wholeslurry, is subsequently subject to fermentation to produce ethanol.

In some embodiments, thin stillage recovered after fermentation of ahydrolysate is used as a substrate for biomethane production. In someembodiments, separated liquid fraction is blended with thin stillagerecovered after fermentation of a hydrolysate and used as a combinedbiomethane substrate.

It will be readily understood that “solid fraction” and “liquidfraction” may be further subdivided or processed. In some embodiments,biomass may be removed from a pretreatment reactor concurrently withsolid/liquid separation. In some embodiments, pretreated biomass issubject to a solid/liquid separation step after it has been unloadedfrom the reactor, typically using a simple and low cost screw presssystem, to generate an solid fraction and a liquid fraction.

As is well known in the art, enzymatic hydrolysis using cellulaseactivity is more efficient where hydrolysis is conducted at lower drymatter content. Higher solids concentration effectively inhibitscellulase catalysis, although the precise reasons for this well knowneffect are not fully understood. See e.g. Kristensen et al. (2009).While the prevailing view in the art is that hydrolysis at the highestpractical dry matter content is advantageous, this is necessarilyassociated with increased enzyme consumption. The same enzymatichydrolysis effects can be achieved using the same enzyme preparations atlower dry matter content with savings of enzyme costs.

One skilled in the art will readily determine, through routineexperimentation, a DM level at which to conduct enzymatic hydrolysisthat is appropriate to achieve given process goals, for any givenbiomass feedstock and enzyme preparation. In some embodiments, it may beadvantageous to conduct hydrolysis at very high DM>20%, notwithstandingsome resulting increase in enzyme consumption. It is generallyconsidered advantageous to conduct hydrolysis at the highest practicabledry matter level, for a variety of reasons including minimizing waterconsumption and sparing energy costs in ethanol distillation, wherehigher sugar concentrations produced by enzymatic hydrolysis at higherdry matter levels result in higher ethanol concentrations, which in turnreduces distillation costs. In some embodiments, enzymatic hydrolysis ofa separated solid fraction or of whole slurry may be conducted at 8% DMor greater, or at 9% DM or greater, or at 10% DM or greater, or at 11%DM or greater, or at 12% DM or greater, or at 13% DM or greater, or at14% DM or greater, or at 15% DM or greater, or at 16% DM or greater, orat 17% DM or greater, or at 18% DM or greater or at 19% DM or greater,or at 20% DM or greater, or at 21% DM or greater, or at 22% DM orgreater, or at 23% DM or greater, or at 25% DM or greater, or at 30% DMor greater, or at 35% DM or greater.

In some embodiments, solid fraction is recovered from solid/liquidseparation of pretreated biomass at 40% DM or greater, but additionalwater content is added so that enzymatic hydrolysis may be conducted atlower DM levels. It will be readily understood that water content may beadded in the form of fresh water, condensate or other process solutionswith or without additives such as polyethylene glycol (PEG) of anymolecular weight or surfactants, salts, chemicals for pH adjustment suchas ammonia, ammonium hydroxide, calcium hydroxide, or sodium hydroxide,anti-bacterial or anti-fungal agents, or other materials.

In order to achieve C5 monomer yields in hydrolysate of at least 55% orgreater, according to methods of the invention, enzymatic hydrolysis isconducted using a variety of enzyme activities. The mixture of enzymesused in enzymatic hydrolysis should comprise at least the followingactivities endoglucanase, exoglucanase, β-glucosidase, endoxylanase, andβ-xylosidase. It will be readily understood by one skilled in the artthat various different enzyme dose levels may be applied, depending onthe dry matter content under which hydrolysis is conducted, the glucanconversion yields desired as process goals, and the hydrolysis timesdesired as process goals. Thus, an enzyme dose level appropriate forhigher dry matter, rapid hydrolysis may be greatly reduced and used in alower dry matter, longer hydrolysis time frame.

As a general rule, C5 monomer yields in hydrolysate of at least 55% orgreater can be achieved relatively quickly, typically in time frames aslow as 24 hours, and generally within the range 18 hours to 60 hours. Itis advantageous to achieve these very high C5 monomer recoveries asquickly as practicable, because once xylan content is substantiallyremoved from undissolved solids, endo- and exo-glucanases arecomparatively less hindered in approach to productive binding. In someembodiments, it can be advantageous to supplement an enzyme mixture withexcess endo- and exo-glucanase activity after high C5 monomer yields areachieved. The hydrolysis time frames over which these high C5 monomeryields can typically be achieved are indicated, for example, in FIG. 11,and as explained in Example 10, showing xylan conversion in whole slurryhydrolysis at 12% DM, where total C5 recovery after pretreatment wasabout 77%. As shown, even at very low enzyme dose levels, C5 monomeryields of at least 55% can be achieved within 40 hours.

Appropriate levels of the various enzyme activities suitable forpracticing methods of the invention so as to achieve C5 monomer yieldsof 55% or greater are typically as follows:

Endoglucanase refers to 4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase alsoknown as β-1,4-glucanase (EC 3.2.1.4). For purposes of defining limitingvalues, endoglucanase activities are determined using the method ofGhose (1987) using hydroxyl-ethyl cellulose (HEC) as substrate andexpressed in nkat/g enzyme preparation. Typically endoglucanase levelsshould be at least 1100 nkat/g glucan at the start of enzymatichydrolysis. Depending on process goals, such as hydrolysis speed,conversion degree and dry matter content, endoglucanase activity levelsmay vary within the range 1100-30000 nkat/g glucan. In some embodiments,the range may be between 1100-2832, or between 1130-1529, or between2317-3852, or between 3000-5120, or between 4000-8000, or between7000-10000, or between 11000-20000.

Exoglucanase refers to 4-β-D-glucan cellobiohydrolase (EC 3.2.1.91). Forpurposes of defining limiting values, exoglucanase activities aredetermined using the method of Bailey and Tahtiharju (2003) using4-methyl-umbelliferyl-β-D-lactoside as substrate and expressed in nkat/genzyme preparation. Typically exoglucanase levels should be at least 280nkat/g glucan at the start of enzymatic hydrolysis. Depending on processgoals, such as hydrolysis speed, conversion degree and dry mattercontent, activity levels may vary within the range 280-20000 nkat/gglucan. In some embodiments, the range may be between 280-690, orbetween 370-560, or between 400-932, or between 700-1240, or between1200-2000, or between 3000-5000.

3-glucosidase refers to β-D-glucoside glucohydrolase (EC 3.2.1.21). Forpurposes of defining limiting values, β-glucosidase activities aredetermined using the method of Berghem and Pettersson (1974) using4-nitrophenyl-β-D-glucopyranoside as substrate and expressed in nkat/genzyme preparation. Typically β-glucosidase levels should be at least3000 nkat/g glucan at the start of enzymatic hydrolysis. Depending onprocess goals, such as hydrolysis speed, conversion degree and drymatter content, activity levels may vary within the range 3000-50000nkat/g glucan. In some embodiments, the range may be between 3000-7500,or between 4000-6010, or between 5000-1000, or between 7000-14000, orbetween 15000-25000.

Endoxylanase refers to 4-β-D-xylan xylanohydrolase (EC 3.2.1.8). Forpurposes of defining limiting values, endoxylanase activities aredetermined using the method of Bailey et al. (1992) using birchwoodxylan as substrate and expressed in nkat/g enzyme preparation. Citratebuffer may be used to adjust pH to an appropriate level for the testedactivity. Typically endoxylanase levels should be at least 1400 nkat/gglucan at the start of enzymatic hydrolysis. Depending on process goals,such as hydrolysis speed, conversion degree and dry matter content,activity levels may vary within the range 1400-70000 nkat/g glucan. Insome embodiments, the range may be between 1400-3800, or between4000-5000, or between 6000-7000, or between 7000-8000, or between9000-12000, or between 11000-15000, or between 15000-20000, or between18000-30000.

β-xylosidase refers to 4-β-D-xylan xylohydrolase (EC 3.2.1.37). Forpurposes of defining limiting values, β-xylosidase activities aredetermined using the method of Poutanen and Puls (1988) usingpara-nitrophenyl-β-D-xylanopyranoside as substrate and expressed innkat/g enzyme preparation. Typically β-xylosidase levels should be atleast 75 nkat/g glucan at the start of enzymatic hydrolysis. Dependingon process goals, such as hydrolysis speed, conversion degree and drymatter content, activity levels may vary within the range 75-124, orbetween 100-300, or between 250-500, or between 400-800, or between700-20000 nkat/g glucan. In some embodiments, the range may be between700-900, or between 800-1400, or between 1100-1700, or between1500-2500, or between 2000-3500, or between 3000-5000, or between4000-10000, or between 8000-20000.

Any of the assays indicated above to be used for activity determinationsmay be modified in appropriate ways, including that samples may beadjusted to appropriate dilution for purposes of measurements. The assaymay be adapted for measurements in representative samples taken from ahydrolysis mixture by comparison with standard curves where knownactivities are added to samples with similar background dry mattercontent.

As will be readily understood by one skilled in the art, the compositionof enzyme mixtures suitable for practicing methods of the invention mayvary within comparatively wide bounds. Suitable enzyme preparationsinclude commercially available cellulase preparations optimized forlignocellulosic biomass conversion. Selection and modification of enzymemixtures during optimization may include genetic engineering techniques,for example such as those described by Zhang et al. (2006) or by othermethods known in the art. Commercially available cellulase preparationsoptimized for lignocellulosic biomass conversion are typicallyidentified by the manufacturer and/or purveyor as such. These aretypically distinct from commercially available cellulase preparationsfor general use or optimized for use in production of animal feed, food,textiles detergents or in the paper industry. In some embodiments, acommercially available cellulase preparation optimized forlignocellulosic biomass conversion is used that is provided by GENENCOR™and that comprises exoglucanases, endoglucanases, endoxylanases,xylosidases, and beta glucosidases isolated from fermentations ofgenetically modified Trichoderma reesei, such as, for example, thecommercial cellulase preparation sold under the trademark ACCELLERASETRIO™. In some embodiments, a commercially available cellulasepreparation optimized for lignocellulosic biomass conversion is usedthat is provided by NOVOZYMES™ and that comprises exoglucanases,endoglucanases, endoxylanases, xylosidases, and beta glucosidases, suchas, for example, the commercial cellulase preparations sold under eitherof the trademarks CELLIC CTEC2™ or CELLIC CTEC3™.

The enzyme activities represented in three commercially availablecellulase preparation optimized for lignocellulosic biomass conversionwere analysed in detail. For each of these commercial cellulasepreparations, levels of twelve different enzyme activities werecharacterized and expressed per gram protein. Experimental details areprovided in Example 8. Results are shown in Table 1. It should be notedthat the assay methods used in this example are not the same as thoserelied upon for determinations of activities herein. These resultsprovide only a generalized, qualitative comparison and should not beviewed as limiting regarding claims to methods of the invention.

TABLE 1 Selected activity measurements in commercial cellulasepreparations optimized for lignocellulosic biomass conversion. ActivityUnit definition CTEC 3 ACTrio CTEC2 Substrate (formation) CBH I 454 ±2.5 U/g 171 ± 0.4 U/g 381 ± 21 U/g MeUmb-3- cellobioside 1 μmole MeUmdequivalent/min CBH II* Not measurable Not measurable Not measurableEndo-1,4-β-glucanase 466 ± 31 U/g 149 ± 21 U/g 173 ± 15 U/g AvicelPH-101 1 μmole glucose equivalent/min. β-glucosidase 3350 ± 75 U/g 891 ±60 U/g 2447 ± 70 U/g Cellobiose 2 μmole glucose/min. (Conversion of 1μmole cellobiose/min) Endo-1,4-β-xylanase 278 ± 10 U/g 799 ± 55 U/g 306± 41 U/g WEAX (medium visc.) 1 μmole glucose equivalent/min.β-xylosidase 279 ± 7.0 U/g 431 ± 22 U/g 87 ± 0.2 U/g WEAX (medium visc.)1 μmole xylose/min. β-L-arabinofuranosidase 20 ± 1.0 U/g 9.4 ± 0.4 U/g12 ± 0.1 U/g WEAX (medium visc.) 1 μmole arabinose/min. Laccase Noactivity No activity No activity Syringaldazine — Amyloglucosidase (AMG)18 ± 3.6 U/g 29 ± 0.1 U/g 18 ± 1.5 U/g Corn starch (soluble) 1 μmoleglucose/min. o-amylase 2.7 ± 0.1 U/g 3.4 ± 0.5 U/g 4.7 ± 1.4 U/g Cornstarch (soluble) 1 μmole glucose equivalent/min. Acetyl xylan esterase3.8 · 10⁻³ ± 3.1 · 10⁻⁴ ± 4.2 · 10⁻³ ± pNP-acetate 1 μmole pNP 9 · 10⁻⁵U/g 1 · 10⁻⁴ U/g 4.2 · 10⁻⁴ U/g equivalent/min. Ferulic acid esterase Noactivity No activity No activity Methyl ferulate —

Enzyme mixtures that are effective to hydrolyse lignocellulosic biomasscan alternatively be obtained by methods well known in the art from avariety of microorganisms, including aerobic and anaerobic bacteria,white rot fungi, soft rot fungi and anaerobic fungi. See e.g. Singhaniaet al. (2010). Organisms that produce cellulases typically secrete amixture of different enzymes in appropriate proportions so as to besuitable for hydrolysis of lignocellulosic substrates. Preferred sourcesof cellulase preparations useful for conversion of lignocellulosicbiomass include fungi such as species of Trichoderma, Penicillium,Fusarium, Humicola, Aspergillus and Phanerochaete.

One fungus species in particular, Trichoderma reesei, has beenextensively studied. Wild type Trichoderma reesei secretes a mixture ofenzymes comprising two exocellulases (cellobiohydrolases) withrespective specificities for reducing and non-reducing ends of cellulosechains, at least five different endocellulases having differingcellulose recognition sites, two B-glucosidases as well as a variety ofendoxylanases and exoxylosidases. See Rouvinen, J., et al. (1990);Divne, C., et al. (1994); Martinez, D., et al. (2008). Commercialcellulase preparations typically also include alpha-arabinofuranosidaseand acetyl xylan esterase activities. See e.g. Vinzant, T., et al.(2001).

An optimized mixture of enzyme activities in relative proportions thatdiffer from the proportions presented in mixtures naturally secreted bywild type organisms has previously been shown to produce higher sugaryields. See Rosgaard et al. (2007). Indeed, it is has been suggestedthat optimizations of enzyme blends including as many as 16 differentenzyme proteins can be advantageously determined separately for anygiven biomass feedstock subject to any given pretreatment. See Billard,H., et al. (2012); Banerjee, G., et al. (2010). As a commercialpracticality, however, commercial enzyme providers typically seek toproduce the smallest practicable number of different enzyme blends, inorder that economies of scale can be obtained in large-scale production.

In some embodiments, it can be advantageous to supplement a commerciallyavailable cellulase preparation optimized for lignocellulosic biomassconversion with one or more additional or supplemental enzymeactivities. In some embodiments, it may be advantageous simply toincrease the relative proportion of one or more component enzymespresent in the commercial preparation. In some embodiments, it may beadvantageous to introduce specialized additional activities. Forexample, in practicing methods of the invention using any given biomassfeedstock, particular unhydrolysed carbohydrate linkages may beidentified that could be advantageously hydrolysed through use of one ormore supplemental enzyme activities. Such unhydrolysed linkages may beidentified through characterization of oligomeric carbohydrates, usingmethods well known in the art, in soluble hydrolysates or in insolubleunhydrolysed residual. Unhydrolysed linkages may also be identifiedthrough comprehensive microarray polymer profiling, using monoclonalantibodies directed against specific carbohydrate linkages, as describedby Nguema-Ona et al. (2012). In some embodiments it can be advantageousto supplement a commercially available cellulase preparation optimizedfor lignocellulosic biomass conversion using any one or more ofadditional endoxylanase, B-glucosidase, mannanase, glucouronidase, xylanesterase, amylase, xylosidase, glucouranyl esterase, orarabinofuranosidase.

In some embodiments, it can alternatively be advantageous to produceenzymes on-site at a lignocellulosic biomass processing facility, asdescribed by Humbird et al. (2011). In some embodiments, a commerciallyavailable cellulase preparation optimized for lignocellulosic biomassconversion may be produced on-site, with or without customizedsupplementation of specific enzyme activities appropriate to aparticular biomass feedstock.

In some embodiments the enzyme mixture may further include any one ormore of mannosidases (EC 3.2.1.25), a-D-galactosidases (EC 3.2.1.22),a-L-arabinofuranosidases (EC 3.2.1.55), a-D-glucuronidases (EC3.2.1.139), cinnamoyl esterases (EC 3.1.1.-), or feruloyl esterases (EC3.1.1.73), acetyl xylan esterases (EC 3.1.1.72); B-1,3 xylosidaseactivity (EC 3.2.1.72); alpha 1,3 and/or alpha 1, 5 arabinofuranosidaseactivity (EC 3.2.1.23); or other activities.

Another startling feature of biomass that has been pretreated bysingle-stage autohydrolysis to very low severity levels is that theconcentrations of pretreatment by-products that serve as inhibitors offermentive organisms are kept to very low levels. As a consequence, itis typically possible to use hydrolysed biomass obtained by methods ofthe invention directly in fermentations, without requirement for anywashing or other de-toxification step.

As is well known in the art, autohydrolysis hydrothermal pretreatmenttypically produces a variety of soluble by-products which act as“fermentation inhibitors,” in that these inhibit growth and/ormetabolism of fermentive organisms. Different fermentation inhibitorsare produced in different amounts, depending on the properties of thelignocellulosic feedstock and on the severity of pretreatment. SeeKlinke et al. (2004). At least three categories of fermentationinhibitors are typically formed during autohydrolysis pretreatment: (1)furans, primarily 2-furfural and 5-HMF (5 hydroxymethylfurfural) whichare degradation products from mono- or oligo-saccharides; (2) monomericphenols, which are degradation products of the lignin structure; and (3)small organic acids, primarily acetic acid, which originate from acetylgroups in hemicelluloses, and lignin. The mixture of differentinhibitors has been shown to act synergistically in bioethanolfermentation using yeast strains, see e.g. Palmquist et al. (1999), and,also, using ethanolic Escherichia coli, see e.g. Zaldivar et al. (1999).In some embodiments, it can be advantageous to subject pretreatedbiomass to flash evaporation, using methods well known in the art, inorder to reduce levels of volatile inhibitors, most notably furfural.Using autohydrolysis with typical strains of biomass feedstocks such aswheat straw, sweet sorghum bagasse, sugar cane bagasse, corn stover, andempty fruit bunches, pretreated to xylan number 10% or higher, in ourexperience only acetic acid and furfural levels are potentiallyinhibitory of fermentive organisms. Where biomass feedstocks arepretreated at DM 35% or higher to xylan number 10% or higher, and wheresolid fraction is subsequently hydrolysed enzymatically at 25% or lowerDM, with added water to adjust DM but without washing steps, furfurallevels in the hydrolysate can typically be kept under 3 g/kg and aceticacid levels beneath 9 g/kg. These levels are typically acceptable foryeast fermentations using specialized strains. During enzymatichydrolysis, some additional acetic acid is released from degradation ofhemicellulose in the solid fraction. In some embodiments, it may beadvantageous to remove some acetic acid content from liquid fractionand/or hydrolysed solid fraction using electrodialysis or other methodsknown in the art.

Different feedstocks can be pretreated using single-stage autohydrolysisto low severity log Ro sufficient to produce pretreated biomass havingxylan number 10% or greater using a variety of different combinations ofreactor residence times and temperatures. One skilled in the art willreadily determine through routine experimentation an appropriatepretreatment routine to apply with any given feedstock, using any givenreactor, and with any given biomass reactor-loading andreactor-unloading system. Where feedstocks are pretreated using acontinuous reactor, loaded by either a sluice-system or a screw-plugfeeder, and unloaded by either a “particle pump” sluice system or ahydrocyclone system, very low severity of 10% or greater xylan numbercan be achieved using typical strains of wheat straw or empty fruitbunches by a temperature of 180° C. and a reactor residence time of 24minutes, or temperatures within the range 175° C. to 185° C. forresidence times within the range 18 to 35 minutes, or temperatureswithin the range 170° C. to 190° C. for residence times within the range13 to 40 minutes. For typical strains of corn stover, sugar canebagasse, and sweet sorghum bagasse, very low severity of 10% or greaterxylan number can typically be achieved using within the range 175° C. to185° C. for residence times within the range 8 to 25 minutes, ortemperatures within the range 170° C. to 190° C. for residence timeswithin the range 6 to 35 minutes. It will be readily understood by oneskilled in the art that residence times and temperatures maybe adjustedto achieve comparable levels of R_(o) severity.

Enzymatic hydrolysis of feedstocks pretreated to xylan number 10% orhigher can typically be conducted at high DM>20% with commerciallyreasonable enzyme consumption, without requirement for specific washingor de-toxification steps, where the solid fraction is pressed to atleast 40% DM, or where dissolved solids content of the solid fraction isreduced by at least 50%.

In some embodiments the enzyme mixture may further include any one ormore of mannosidases (EC 3.2.1.25), a-D-galactosidases (EC 3.2.1.22),a-L-arabinofuranosidases (EC 3.2.1.55), a-D-glucuronidases (EC3.2.1.139), cinnamoyl esterases (EC 3.1.1.-), or feruloyl esterases (EC3.1.1.73), acetyl xylan esterases (EC 3.1.1.72); B-1,3 xylosidaseactivity (EC 3.2.1.72); alpha 1,3 and/or alpha 1, 5 arabinofuranosidaseactivity (EC 3.2.1.23); or other activities.

One skilled in the art will readily determine an appropriate dose levelof any given enzyme preparation to apply, and appropriate pH andtemperature conditions as well as an appropriate duration for enzymatichydrolysis. As mentioned previously, duration of hydrolysis may varydepending on process goals. Longer hydrolysis leads to better ultimateglucose conversion yields, but imposes greater capital and operatingcosts at production scale. Hydrolysis duration in some embodiments is atleast 24 hours, or at least 36 hours, or at least 48 hours, or at least64 hours, or at least 72 hours, or at least 96 hours, or for a timebetween 24 and 150 hours. It is generally advantageous to maintain lowerenzyme dose levels, so as to minimize enzyme costs. In some embodiments,it can be advantageous to use a high enzyme dose. In practicing methodsof the invention, one skilled in the art can determine an economicoptimisation of enzyme dose in consideration of relevant factorsincluding local biomass costs, market prices for product streams, totalplant capital costs and amortization schemes, and other factors. Inembodiments where a commercially available cellulase preparationoptimized for lignocellulosic biomass conversion is used, a general doserange provided by manufacturers can be used to determine the generalrange within which to optimize.

In some embodiments, after a separated solid fraction has beenenzymatically hydrolysed to a desired degree of conversion, the liquidfraction, which has been maintained in C5 bypass, is mixed with thehydrolysate mixture for post-hydrolysis. In some embodiments, all of therecovered liquid fraction may be added at one time, while in otherembodiments, some component of the liquid fraction may be removed and/orliquid fraction may be added incrementally. In some embodiments, priorto mixing with liquid fraction, the solid fraction is hydrolysed to atleast 50%, or at least 55%, or at least 60% cellulose conversion,meaning that at least the specified theoretical yield of glucosemonomers is obtained in the hydrolysate. A substantial portion ofxylo-oligomers present in liquid fraction can typically be hydrolysed toxylose monomers by action of xylanase and other enzymes that remainactive within the hydrolysate mixture. In some embodimentspost-hydrolysis is conducted for at least 6 hours, or for a time between15 and 50 hours, or for at least 24 hours. In some embodiments, at least60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%,or at least 85%, or at least 90% by mass of xylo-oligomers present inthe liquid fraction are hydrolysed to xylose monomers duringpost-hydrolysis by action of xylanase and other enzymes that remainactive within the hydrolysate mixture. In some embodiments, the liquidfraction is mixed with hydrolysate directly, without further addition ofchemical additives. In some embodiments, some components of liquidfraction such as acetic acid, furfural or phenols may be removed fromliquid fraction prior to mixing with hydrolysate.

In some embodiments, enzymatic hydrolysis of the solid fraction and/orpost-hydrolysis of the liquid fraction may be conducted as asimultaneous saccharification and fermentation (SSF) process. As is wellknown in the art, when SSF can be conducted at the same temperature asthat which is optimal for enzymatic hydrolysis, enzyme consumption canbe minimized because a fermentive organism introduced during the courseof enzymatic hydrolysis consumes glucose and xylose monomers and therebyreduces product inhibition of enzyme catalyzed reactions. In someembodiments, post-hydrolysis is only conducted after the fiber fractionhas been hydrolysed, without addition of fermentive organism, to atleast 60% cellulose conversion. In some embodiments SSF can be conductedafter an initial period of enzymatic hydrolysis, that is a fermentiveorganisms added after an initial period of enzymatic hydrolysis, andboth fermentation and hydrolysis continued, optionally at a temperaturethat is not optimal for enzymatic hydrolysis.

Where biomass feedstocks such as typical strains of wheat straw, sugarcane bagasse, sweet sorghum bagasse, corn stover or empty fruit bunchesare pretreated at 35% or greater DM by single-stage autohydrolysis toseverity log Ro sufficiently low so as to produce pretreated biomasshaving xylan number 10% or greater, where solid fraction of thepretreated biomass is obtained having at least 40% DM or having at least50% removal of dissolved solids, where solid fraction is subsequentlysubject to enzymatic hydrolysis at DM between 15 and 27% using acommercially available cellulase preparation optimized forlignocellulosic biomass conversion, where enzymatic hydrolysis isconducted for at least 48 hours, where liquid fraction is added to thesolid fraction hydrolysate after at least 50% glucose conversion hasbeen obtained, and where the added liquid fraction is subject topost-hydrolysis for a period of at least 6 hours, it is typicallypossible to achieve C5 monomer concentrations in the combined C5/C6hydrolysate that correspond to C5 monomer yields of 60% or greater ofthe theoretical maximal C5 monomer yield.

In some embodiments, a combined C5/C6 hydrolysate can be directlyfermented to ethanol using one or more modified yeast strains.

FIG. 9 shows a process scheme for one embodiment. As shown, softlignocellulosic biomass is soaked, washed or wetted to DM 35% orgreater. The biomass is pretreated at pH within the range of 3.5 to 9.0using pressurized steam in single-stage autohydrolysis to a severitycharacterized by xylan number 10% or greater. The pretreated biomass issubject to solid/liquid separation producing a liquid fraction and asolid fraction having DM content 40% or greater. The solid fraction isadjusted to an appropriate DM content then subject to enzymatichydrolysis at DM content 15% or greater to a degree of celluloseconversion 60% or greater. The separated liquid fraction is subsequentlymixed with the hydrolysed solid fraction and subject to post-hydrolysis,whereby a substantial quantity of xylo-oligomers present in the liquidfraction are hydrolysed to monomeric xylose. After the end of hydrolysisand post-hydrolysis as described, the C5 monomer yield is typically atleast 60% while the cellulose conversion is similarly at least 60%.

In alternative embodiments, the pretreated biomass is subject toenzymatic hydrolysis as a whole slurry. In still other embodiments, aliquid fraction is separated and a solid fraction subject to enzymatichydrolysis and subsequent fermentation. In some embodiments, followingethanol recovery from such a fermentation, the remaining thin stillagecan be blended with separated liquid fraction and used as biomethanesubstrate.

EXAMPLES Example 1 “Xylan Number” Characterization of Solid Fraction asa Measure of Pretreatment Severity

Wheat straw (WS), corn stover (CS), Sweet sugarcane bagasse (SCB) andEmpty Fruit Bunches (EFB) were soaked with 0-10 g acetic acid/kg drymatter biomass, pH>4.0, prior to pretreatment at 35-50% dry matter About60 kg DM/h biomass was pretreated at temperatures from 170-200° C. witha residence time of 12-18 minutes. The biomass was loaded into thereactor using a sluice system and the pretreated material unloaded usinga sluice system. The pressure within the pressurized pretreatmentreactor corresponded to the pressure of saturated steam at thetemperature used. The pretreated biomass was subject to solid/liquidseparation using a screw press, producing a liquid fraction and a solidfraction having about 30% dry matter. The solid fraction was washed withabout 3 kg water/kg dry biomass and pressed to about 30% dry matteragain. Details concerning the pretreatment reactor and process arefurther described in Petersen et al. (2009).

Raw feedstocks were analysed for carbohydrates according to the methodsdescribed in Sluiter el al. (2005) and Sluiter et al. (2008) using aDionex Ultimate 3000 HPLC system equipped with a Rezex Monossacharide H+column from Phenomenex. Samples of liquid fraction and solid fractionwere collected after three hours of continuous pretreatment and sampleswere collected three times over three hours to ensure that a sample wasobtained from steady state pretreatment. The solid fractions wereanalysed for carbohydrates according to the methods described in Sluiteret al. (2008) with an Ultimate 3000 HPLC system from Dionex equippedwith a Rezex Monossacharide H+ Monosaccharide column. The liquidfractions were analysed for carbohydrates and degradation productsaccording to the methods described in Sluiter et al. (2006) with anUltimate 3000 HPLC system from Dionex equipped with a RezexMonossacharide H+ Monosaccharide column. Degradation products in thesolid fraction were analysed by suspension of the solid fraction inwater with 5 mM sulphuric acid in a ratio of 1:4 and afterward analysedaccording to the methods described in Sluiter et al. (2006) with anUltimate 3000 HPLC system from Dionex equipped with a RezexMonossacharide H+ column. The dry matter content and the amount ofsuspended solids was analysed according to the methods described inWeiss et al. (2009). Mass balances were set up as described in Petersenet al. (2009) and cellulose and hemicellulose recoveries weredetermined. The amount of sugars which were degraded to 5-HMF orfurfural and the amount of acetate released from hemicelleulose duringpretreatment per kg of biomass dry matter was quantified as well,although loss of furfural due to flashing is not accounted for.

The severity of a pretreatment process is commonly described by aseverity factor, first developed by Overend et al. (1987). The severityfactor is typically expressed as a log value such thatlog(R₀)=t*eksp((T−Tref)/14.75), where R₀ is the severity factor, t isthe residence time in minutes, T is the temperature and T_(ref) is thereference temperature, typically 100° C. The severity factor is based onkinetics of hemicellulose solubilisation as described by Belkecemi etal. (1991), Jacobsen and Wyman (2000) or Lloyd et al. (2003). Theseverity of a pretreatment is thus related to residual hemicellulosecontent remaining in the solid fraction after pretreatment.

Solid fractions prepared and washed as described were analysed for C5content according to the methods described by Sluiter et al. (2008) witha Dionex Ultimate 3000 HPLC system equipped with a Rezex MonossacharideH+ column from Phenomenex. The xylan content in the solid fractionproduced and washed as described above is linearly depended upon theseverity factor for soft lignocellulosic biomasses such as for examplewheat straw, corn stover of EFB when pretreating by hydrothermalautohydrolysis. The definition of severity as the xylan content in asolid fraction prepared and washed as described above is transferablebetween pretreatment setups. Xylan number is the measured xylan contentin the washed solid fractions, which includes some contribution fromsoluble material. The dependence of xylan number on pretreatmentseverity log(R_(o)) is shown in FIG. 1 for wheat straw, corn stover,sugarcane bagasse and empty fruit bunches from palm oil processing.

As shown, there exists a clear, negative linear correlation betweenxylan number and pretreatment severity for each of the tested biomassfeedstocks pretreated by single-stage autohydrolysis.

The xylan content of undissolved solids in the experiments was alsocalculated as the total xylan content in fibre fraction from which issubtracted the content of dissolved xylan in the liquid between thefibres (oligomers and monomers).[Solid Xylan in fibres](wt-%)=[Total xylan in fibrefraction](wt-%)−[Dissolved xylan in fibre fraction](wt-%)

The dissolved xylan content is calculated by [(dissolved solids/totalsolids) as wt % in fibre fraction]×[dissolved xylan concentration inliquid fraction].

Calculated xylan content of undissolved solids in wt % is shown as afunction of Xylan number in FIG. 2 for pretreated wheat straw (PWS),corn stover (PCS), sugarcane bagasse (SCB) and empty fruit bunches fromoil palm (PEFB).

Example 2 C5 Recovery as a Function of Pretreatment Severity

Biomass feedstocks were pretreated and samples characterized asdescribed in example 1. FIG. 3 shows the C5 recoveries(xylose+arabinose) as a function of xylan number for experiments wherewheat straw was pretreated by single-stage autohydrolysis. C5 recoveriesare shown as water insoluble solids (WIS), water soluble solids (WSS)and total recovery. As shown, C5 recovery as both water insoluble andwater soluble solids increases as xylan number increases. As xylannumber increases over 10%, C5 recovery as water soluble solidsdiminishes while C5 recovery as water insoluble solids continues toincrease

Typical strains of wheat straw tested contained about 27% hemicelluloseon dry matter basis prior to pretreatment. FIG. 4 shows total C5recovery after pretreatment as a function of Xylan number for wheatstraw, corn stover, sugarcane bagasse and EFB pretreated byautohydrolysis. Typical strains of corn stover, sweet sugarcane bagasseand EFB tested contained about 25%, 19% and 23% respectively of C5content on dry matter basis prior to pretreatment. As shown, for allfeedstocks, total C5 recovery after pretreatment is dependent uponpretreatment severity as defined by the xylan number of pretreatedbiomass. As shown, where 90% of C5 content recovered after pretreatmentcan be fully hydrolysed to C5 monomer, at least 60% final C5 monomeryield after enzymatic hydrolysis can typically be expected wherepretreatment severity is characterized by producing a xylan number of10% or higher.

Example 3 Production of Degradation Products that Inhibit Enzymes andYeast Growth as a Function of Pretreatment Severity

Biomass feedstocks were pretreated and samples characterized asdescribed in example 1. FIG. 5 shows the dependence of acetic acidrelease and production of furfural and 5-hydroxy-methyl-fufural (5-HMF)as a function of xylan number for experiments where wheat straw waspretreated by single-stage autohydrolysis. As shown, production of thesedegradation products, which are well known to inhibit fermentive yeastand which in some cases also inhibit cellulase enzymes, exhibits anexponential increase at xylan numbers lower than 10%. At xylan number10% and higher, the levels of furfural and acetic acid fall withinranges that permit fermentation of pretreated biomass withoutrequirement for de-toxification steps. In the case of acetic acid,levels are further increased during enzymatic hydrolysis of biomasspretreated to xylan number 10% and higher, although typically to levelsthat are well tolerated by yeast modified to consume both C5 and C6sugars.

Example 4 Inhibition of Cellulase Enzymes by Material Remaining in SolidFraction as a Function of DM % of Solid Fraction

Experiments were conducted in a 6-chamber free fall reactor working inprinciple as the 6-chamber reactor described and used in WO2006/056838.The 6-chamber hydrolysis reactor was designed in order to performexperiments with liquefaction and hydrolysis at solid concentrationsabove 20% DM. The reactor consists of a horizontally placed drum dividedinto 6 separate chambers each 24 cm wide and 50 cm in height. Ahorizontal rotating shaft mounted with three paddles in each chamber isused for mixing/agitation. A 1.1 kW motor is used as drive and therotational speed is adjustable within the range of 2.5 and 16.5 rpm. Thedirection of rotation is programmed to shift every second minute betweenclock and anti-clock wise. A water-filled heating jacket on the outsideenables control of the temperature up to 80° C.

The experiments used wheat straw, pretreated by single-stageautohydrolysis using the system described in example 1. The biomass waswetted to a DM of >35% and pretreated at pH>4.0 by steam to severity logRo approximately 3.7, producing pretreated material having xylan number10.5%. The pretreatment was conducted in the Inbicon pilot plant inSkærbæk, Denmark. The biomass was loaded into the pretreatment reactorusing a sluice system and the pretreated biomass removed from thereactor using a sluice system. The pretreated biomass was, in somecases, subject to solid/liquid separation using a screw press, producinga liquid fraction and a solid fraction. The solid fraction had a DMcontent of about 30%, contained the majority of initial cellulose andlignin, part of the hemicellulose and a total of about 25% of thedissolved solids.

The chambers of the 6 chamber reactor were filled with either totalpretreated biomass comprising all dissolved and undissolved solids orpressed solid fraction comprising about 25% of total dissolved solids.Dry matter content was adjusted to 19% DM. The pretreated biomass wasthen hydrolyzed at 50° C. and pH 5.0 to 5.3 using 0.08 ml CTec2™ fromNovozymes/g glucan or 0.2-0.3 ml Accellerase TRIO™ from Dupont,Genencor/g glucan. These dose levels of these commercially availablecellulase preparations optimized for lignocellulosic biomass conversionwere well within the range suggested by the manufacturers. Enzymatichydrolysis experiments were conducted for 96 hours at a mixing speed of6 rpm.

It can be shown that the enzyme activities in the experiment withAccellerase TRIO as measured as described herein were initially withinthe range exoglucanase 280-5000 nkat/g glucan, endoglucanase 1100-20000nkat/g glucan, β-glucosidase 3000-25000 nkat/g glucan, endoxylanase1400-30000 nkat/g glucan, β-xylosidase 75-25000 nkat/g glucan nkat/gglucan.

FIG. 6 shows cellulose conversion after enzymatic hydrolysis under theseconditions as a function of % dissolved solids removed prior toenzymatic hydrolysis. As shown, removal of 75% dissolved solids at theseenzyme dose levels improves cellulose conversion by 10-20% in absoluteterms. Thus, in cases where enzymatic hydrolysis is to be conductedusing a separated solid fraction, it is advantageous to press the solidfraction to DM content at least 40% or to otherwise reduce dissolvedsolids content by at least 50% prior to enzymatic hydrolysis, since thiswill typically provide improved enzyme performance.

Example 5 Sugar Content and Hydrolysis of Liquid Fraction from BiomassPretreated to Xylan Number >10%

Wheat straw, corn stover, and sugar cane bagasse were pretreated toseverity log Ro 3.63, producing pretreated wheat straw (WS) having xylannumber 11.5%, to log Ro 3.51 producing pretreated sugar cane bagasse(SCB) having xylan number 12.3% and to log Ro 3.35, producing pretreatedcorn stover (CS) having xylan number 15.5%. The pretreated feedstockswere subject to solid/liquid separation to produce a liquid fraction anda solid fraction, as described in example 4. The liquid fractions wereanalysed for carbohydrates and degradation products according to themethods described in (Sluiter, Hames et al. 2005) using a DionexUltimate 3000 HPLC system equipped with a Rezex Monosaccharide column.Table 2 shows the sugar content of liquid fractions expressed as apercent of DM content broken down into categories of oligomeric andmonomeric glucose/glucan, xylose/xylan and arabinose/arabinan. As shown,while some glucose content is present in both monomeric and oligomericform, the bulk of the sugar content is oligomeric xylan. Thepredominance of xylan oligomers in liquid fraction obtained usingautohydrolysis is in noted contrast with the liquid fraction obtainedusing dilute acid pretreatment. In biomass pretreated by dilute acidhydrothermal pretreatment, the liquid fraction is typically hydrolysedto monomeric constituents by actions of the acid catalyst.

TABLE 2 Sugar content of liquid fractions in biomass pretreated to xylannumber >10%. Oligomeric Monomeric Oligomeric Monomeric OligomericMonomeric Other glucan glucose xylan xylose arabinan arabinose DM WS5.5% 2.1% 40.4% 8.6% 1.1% 4.8% 37% SCB 8.2% 3.1% 39.1% 8.7% 0.7% 3.1%37% SC 6.2% 1.9% 37.0% 5.3% 2.8% 3.9% 43%

The liquid fraction from pretreated wheat straw was furthercharacterized by HPLC analysis using a Thermo Scientific DionexCarboPac™ PA200 column using a modular Dionex ICS-5000 chromatographicsystem. The analytes were separated using NaOH/NaOAc-gradient conditionsand measured by integrated and pulsed amperometric detection (IPAD)using a gold electrode. FIG. 7 shows an HPLC chromatogram in which theelution profile of xylobiose (X₂), xylotriose (X₃), xylotetraose (X₄),xylopentaose (X₅), and xylohexaose (X₆) standards is super-imposed asthe upper trace over the lower trace, which depicts the elution profileof liquid fraction. As shown, liquid fraction of the autohydrolysedbiomass contains a mixture comprising a small amount of xylose monomerand comparatively larger amounts of xylobiose (X₂), xylotriose (X₃),xylotetraose (X₄), xylopentaose (X₅), and xylohexaose (X₆), along withother materials.

Example 6 Enzymatic Hydrolysis of Solid Fraction and Addition of LiquidFraction after the Fibre Hydrolysis from Biomass Pretreated to XylanNumber >10% and Pressed to >40% DM Followed by Post Hydrolysis.

Experiments were conducted in a 6-chamber free fall reactor as describedin example 4.

The experiments used wheat straw, corn stover, or sugar cane bagassepretreated by single-stage autohydrolysis to severity log Ro betweenabout 3.19 and 3.73 to produce pretreated biomass having xylan numbersranging from 11.5 to 15.6%. The biomass was cut and wetted to a DMof >35% and pretreated by steam at 170-190° C. for 12 min. Thepretreatment was conducted in the Inbicon pilot plant in Skærbæk,Denmark. The pretreated biomass was subject to solid/liquid separationusing a screw press to produce a solid fraction having >40% DM. Theliquid fraction was saved (C5 bypass) so as to be subsequently added tothe hydrolysate (posthydrolysis).

The chambers of the 6 chamber reactor were filled with about 10 kgpressed pretreated biomass solid fraction and adjusted by water additionto 19-22% DM. The pretreated solid fraction was hydrolyzed at 50° C. andpH 5.0 to 5.3 u sing ACCELLERASE TRIO™ from GENENCOR-DuPONT. The mixingspeed was 6 rpm. The hydrolysis experiments were run for 96 hours andafterwards the saved liquid fraction (C5 bypass) was added and posthydrolysis was run for 48 hours at 50° C. and pH 5.0 to 5.3.

HPLC samples were taken daily to follow the conversion of cellulose andhemicellulose and analysed for glucose, xylose and arabinose using aDionex Ultimate 3000 HPLC system equipped with a Rezex Monosaccharidecolumn with quantification through use of external standard.

FIG. 8 shows hydrolysis data for conversion of hemicellulose withaddition of liquid fraction after 96 hours hydrolysis of solid fractionusing sugar cane bagasse pretreated to xylan number 12.3% and hydrolysedusing 0.3 ml Accellerase Trio™ (Genencor) per g glucan. Shown is atypical hydrolysis profile. C5 monomer recovery is expressed as apercent of theoretical yield from the material present in the hydrolysisreaction. Most of the hemicellulose within the solid fraction has beenconverted to monomeric sugars within the first 24 hours in hydrolysis ofthe solid fraction. Addition of liquid fraction after 96 hours increasesthe theoretical potential yield, which explains the drop in C5conversion observed just after liquid fraction is added. Within thefirst 24 hours most of the C5 from liquid fraction is converted tomonomers. Comparing the C5 conversion just before liquid fraction isadded with the end point of the hydrolysis, it is possible to calculatethe C5 conversion in the liquid fraction as 90% when using sugar canebagasse under these conditions.

Table 3 shows hydrolysis data for different biomasses pretreated underdifferent circumstances and hydrolysed using different dose levels of acommercially available cellulase preparation optimized forlignocellulosic biomass conversion, Accellerase Trio™ (Genencor). Allenzyme dose levels used were within the range suggested by themanufacturer. As shown, using single-stage autohydrolysis and enzymatichydrolysis with C5 bypass and post-hydrolysis, C5 monomer yields of 60%or greater can be achieved using manufacturers' recommended doses ofcommercially available cellulase preparations optimized forlignocellulosic biomass conversion while still achieving celluloseconversion of 60% or greater.

It can be shown that the enzyme activities in the experiments referredto in Table 3 with Accellerase TRIO measured as described herein wereinitially within the range exoglucanase 280-5000 nkat/g glucan,endoglucanase 1100-20000 nkat/g glucan, β-glucosidase 3000-25000 nkat/gglucan, endoxylanase 1400-30000 nkat/g glucan, β-xylosidase 75-25000nkat/g glucan nkat/g glucan.

TABLE 3 Hydrolysis yields using very low severity single-stageautohydrolysis with C5 bypass and post-hydrolysis. WS SCB SCB CS CS EFBDry matter after soaking [wt %] 40% 39% 39% 40% 40% 39% Residence time[min] 12.0  12.0  12.0  12.0  12.0  12.0  Temperature [° C.] 183.0 182.7  182.7  174.5  174.5  185.2  Pretreatment severity [logRo]  3.52 3.51  3.51  3.27  3.27  3.58 C5 recovery from pretreatment [%] 74% 87%87% 88% 88% 84% Xylan number 11.5%   12.3%   12.3%   15.6%   15.6%  15.5%   Enzyme dosage [mL Ac. TRIO/g glucan] 0.2 0.3 0.3 0.3 0.2 0.4 %TS in fiber hydrolysis 22% 22% 22% 19% 22% 22% Cellulose conversionafter hydrolysis 78% 64% 66% 68% 58% 69% (96 h) Hemicellulose conversion(C5 recovery) 80% 73% 73% 61% 61% 75% after hydrolysis (96 h) % TS insecond hydrolysis 18% 17% 17% 16% 18% 18 Cellulose conversion after posthydrolysis 78% 65% 67% 67% 61% 72% (144 h) Hemicellulose conversion (C590% 79% 78% 71% 68% 83% recovery) after post hydrolysis (144 h) Overallcellulose conversion 78% 65% 67% 67% 61% 72% Overall C5 monomer yield67% 69% 68% 63% 60% 70%

Example 7 Co-Fermentation to Ethanol of C5 and C6 Sugars in CombinedHydrolysate by Modified Yeast

As an example on the use of a hydrolysate produced from softlignocellulosic biomass (in this case wheat straw) prepared bysingle-stage autohydrolysis pretreatment to a xylan number >10%, FIG. 9shows data for a fermentation performed without detoxification or anyother process steps before fermentation with GMO yeast able to convertboth C5 and C6 sugars (strain V1 from TERRANOL™). The solid fractionfrom pretreated wheat straw separated as described in example 4 washydrolyzed using Cellic Ctec2™ from Novozymes and then combined withsaved liquid fraction and used without any de-toxification to removefermentation inhibitors.

The hydrolysate was adjusted to pH 5.5 with KOH pellets beforefermentation and supplemented with 3 g/L urea. The fermentation wasconducted as a batch fermentation. The initial cell concentration in thereactor was 0.75 g dw/L. The fermentations were controlled at pH 5.5using automatic addition of 10% NH3. The temperature was kept at 30′Cand the stirring rate was 300 rpm. As shown, glucose and xylose arereadily consumed and ethanol readily produced, notwithstanding thepresence of acetic acid, furfural and other compounds that wouldtypically prove inhibitory at higher levels of pretreatment severity.

Example 8 Experimental Determination of Activity Levels in CommercialCellulase Preparations

Commercial preparations of ACCELLERASE TRIO™ from GENENCOR™ and CELLICCTEC2™ and CELLIC CTEC3™ from NOVOZYMES™ were diluted so that the enzymepreparations would have equivalent density, meaning that equivalentsized aliquots had equivalent mass. Equivalent volumes of diluted enzymepreparations were added and assay determinations made in duplicate ortriplicate.

Assay of CBHI (exocellulase) activity was conducted in 50 mM NaOACbuffer at pH 5, 25° C., for 25 minutes. Activity was determined intriplicate by following continuous rate of 4-Methylumbelliferon release(Abs: 347 nm) from the model substrate4-methylumbelliferyl-β-cellobioside. Activity unit was 1 umole MeUmbequivalent/minute. Enzyme preparation concentrations were 0.16, 0.14,0.17 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 0.5 mg/ml.

Assay of Endo-1,4-β-glucanase activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 60 minutes. Activity was determined in triplicate by following absorbance change associated with generation ofreducing ends from the model substrate Avicel PH-101. Activity unit was1 μmole glucose equivalent/min. Enzyme preparation concentrations were0.80, 0.67, 0.79 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays.Substrate concentration was 80 mg/ml.

Assay of β-glucosidase activity was conducted in 50 mM NaOAC buffer, pH5; 50° C., for 20 minutes. Activity was determined in triplicate byfollowing absorbance change associated with release of glucose frommodel substrate cellobiose. Activity unit was 2 μmole glucose/min.Enzyme preparation concentrations were 0.1, 0.12, 0.12 mg/mlrespectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 1.7 mg/ml.

Assay of Endo-1,4-β-xylanase activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 60 minutes. Activity was determined in triplicate by following absorbance change associated with generation ofreducing ends from the model substrate water extractable arabinoxylan.Activity unit was 1 μmole glucose equivalent/min. Enzyme preparationconcentrations were 1.12, 0.97, 1.12 mg/ml respectively for CTEC3,ACTrio, and CTEC2 assays. Substrate concentration was 10 mg/ml.

Assay of β-xylosidase activity was conducted in 50 mM NaOAC buffer, pH5; 50° C., for 60 minutes. Activity was determined in duplicate byfollowing release of xylose associated with hydrolysis of the modelsubstrate water extractable arabionxylan. Activity unit was 1 μmolexylose/min. Enzyme preparation concentrations were 1.12, 0.97, 1.12mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 10 mg/ml.

Assay of β-L-arabinofuranosidase activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 60 minutes. Activity was determined intriplicate by following release of arabinoase associated with hydrolysisof the model substrate water extractable arabionxylan. Activity unit was1 μmole arabinose/min. Enzyme preparation concentrations were 1.12,0.97, 1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays.Substrate concentration was 10 mg/ml.

Assay of Amyloglucosidase (AMG) activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 80 minutes. Activity was determined intriplicate by following absorbance change associated with glucoserelease from the model substrate soluble corn starch. Activity unit was1 μmole glucose/min. Enzyme preparation concentrations were 1.12, 0.97,1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 10 mg/ml.

Assay of a-amylase activity was conducted in 50 mM NaOAC buffer, pH 5;50° C., for 60 minutes. Activity was determined in triplicate byfollowing absorbance change associated with generation of reducing endsfrom the model substrate soluble corn starch. Activity unit was 1 μmoleglucose equivalent/min. Enzyme preparation concentrations were 1.12,0.97, 1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays.Substrate concentration was 10 mg/ml.

Assay of acetyl xylan esterase activity was conducted in 100 mMSuccinate buffer, pH 5; 25° C., for 25 minutes. Activity was determinedin triplicate by following continuous rate of 4-Nitrophenyl release(Abs: 410 nm) from the model substrate 4 4-Nitrophenyl acetate. Activityunit was 1 μmole pNP equivalent/min. Enzyme preparation concentrationswere 0.48, 0.42, 0.51 mg/ml respectively for CTEC3, ACTrio, and CTEC2assays. Substrate concentration was 10 mg/ml.

Results of the activity determinations are shown in Table 1.

These results provide a qualitative comparison between the enzymepreparations, but are in most cases not conducted according to themethods used to determine nkat values for enzyme activities for purposesof claims herein.

Example 9 Identification of Enzyme Activities Important for AchievingHigh C5 Monomer Yield in Enzymatic Hydrolysis of Feedstock Pretreated byLow Severity, Single Stage Autohydrolysis

Wheat straw was pretreated as described in example 4 to severity log Ro3.52 (183° C. for a residence time of 12 minutes) to produce apretreated biomass having xylan number 13.5%, with approximately 7.8% byweight xylan remaining in undissolved solids, as estimated from FIG. 2.Glucan recovery from pretreatment was 100%. Xylan recovery frompretreatment was 77%.

Cellulase activity measurements in Filter Paper Units (FPU) weredetermined for three separate commercially available cellulase enzymepreparations ACCELLERASE TRIO™ from GENENCOR™, CELLIC CTEC3™ fromNOVOZYMES™, and a mixture of CELLUCLAST and NOVOZYME 188 from NOVOZYMES™mixed in the ratio by weight 1:0.2 respectively. FPU activities weredetermined by the method of Ghose (1987) and found to be 179 FPU/genzyme preparation for CTEC3, and 60 FPU/g enzyme preparation forCELLUCLCAST/188.

Hydrolysis experiments were conducted essentially as described inexample 6, except that initial dry matter content was 22% DM, 1 wt %polyethylene glycol (PEG) was added, the initial hydrolysis of solidfraction was conducted for 94 hours, posthydrolysis with added liquidfraction (C5 bypass) was conducted for 50 hours, and the enzyme used waseither CTEC3, ACTRIO, or CELLUCLAST/188 applied at an equivalent dose inFPU/g glucan of 14.3 FPU/g glucan.

The actual dose of enzymes applied was CTEC3 0.08 g/g glucan, AcTRIO0.24 g/g glucan, CELLUCLAST/188 0.22 g/g glucan.

Enzyme activities in nkat/g glucan, to be measured as described herein,that were used in the experiment were estimated to be as follows:

Exogluc endogluc beta-gluc xylanase xylosidase Trio 685a 2829b 8016c12024c 267d Celluc/188+ 477  2860  3372  2200 51 CTEC3 607a 2949e10046f   N/A N/A +based on values reported by Juhasz et al. (2005)ameasured using 4-methylumbelliferyl-beta-cellobioside as substratebbased on ACCELLERASE TRIO product information sheet reporting minimalendoglucanase value using carboxymethylcellulose (CMC) as substrate andassuming that the corresponding value for hydroxyethylcellulose will beapproximately 0.35 times the CMC value, such as reported for example byDori et al. (1995) cbased on ACCELLERASE TRIO product information sheetreporting minimal values dbased on alternatively measured ratio ofxylosidase activity comparing Trio:Celluclast of 3.86:1 per g enzymepreparation. ebased on alternatively measured ratio of endoglucanaseactivity comparing CTEC3:TRIO of 3.12:1 per g enzyme preparation. fbasedon alternatively measured ratio of beta-glucosidase activity comparingCTEC3:TRIO of 3.76:1 per g enzyme preparation.

It can be shown that the enzyme activities in these experiments measuredas described herein were initially within the range exoglucanase280-1240 nkat/g glucan, endoglucanase 1100-8000 nkat/g glucan,β-glucosidase 3000-15000 nkat/g glucan, endoxylanase 9000-30000 nkat/gglucan, β-xylosidase 75-1400 nkat/g glucan nkat/g glucan.

FIG. 11 shows cellulose conversion as a function of time in the variousreaction chambers. The cellulose conversion is determined as the glucoseconcentration divided by the theoretical glucose potential at the momentthe sample was taken. When the C5 bypass is added the glucose potentialchanges as the bypass contains a small amount of glucose oligomers whichare not digested resulting in an overall conversion decrease. As shown,the cellulose conversion kinetics are equivalent for the CTEC and TRIOchambers, but not for the CELLUCLAST/188 chamber. This is attributed toappreciably lower levels of xylanase and xylosidase activity.

FIG. 12 shows corresponding xylan conversion as a function of time. Thexylan conversion is calculated in the same way as for the celluloseconversion, but as the C5-bypass contains a large amount of xyloseoligomers the conversion initially dramatically drops when the C5-bypassis added to the hydrolysate. As shown, the xylan conversion kinetics areequivalent for the CTEC and TRIO chambers, but not for theCELLUCLAST/188 chamber. This is again attributed to appreciably lowerlevels of xylanase and xylosidase activity.

Where xylan recovery from pretreatment was 77%, the xylan recoveriesshown in FIG. 12 correspond to very high final C5 monomer recovery inthe hydrolysate, for example, 80% conversion in FIG. 12 corresponds to(0.80)*(0.77)=61.6% C5 monomer recovery.

Glucan and xylan contents are determined as described in Sluiter, A., B.Hames, et al. (2005). Determination of Sugars, Byproducts, andDegradation Products in Liquid Fraction Process Samples, NREL—BiomassProgram and in Sluiter, A., B. Hames, et al. (2006). Determination ofStructural Carbohydrates and Lignin in Biomass, NREL—Biomass Program.

Example 10 Dilution to Lower Dry Matter Permits Equivalent ConversionYields at Lower Enzyme Dose Using Whole Slurry

In the experiments described in example 9, two chambers of the6-chambered hydrolysis reactor were used to compare hydrolysis of wholeslurry, in which the liquid fraction separated from solid fraction afterpretreatment was not saved as bypass to be later added to thehydrolysate, but was instead blended back with solid fraction andhydrolysed at the same time. The whole slurry was diluted to 12% DM,which renders the concentration of inhibitory dissolved substances to beapproximately equivalent with that achieved in the reactions describedin example 9, where dissolved solids were removed and held separate (C5bypass) from hydrolysis of the solid fraction at 22% DM. CTEC3 andACTRIO were used as enzyme preparation and applied at a lower dose of10.7 FPU/g glucan.

Enzyme activities in nkat/g glucan, to be measured as described herein,that were used in the experiment were estimated (based on the valuesgiven in example 9) to be as follows:

Exogluc endogluc beta-gluc xylanase xylosidase Trio 514 2122 6012 9018200 CTEC3 455 2212 7535 N/A N/A

It can be shown that the enzyme activities in these experiments measuredas described herein were initially within the range exoglucanase280-1240 nkat/g glucan, endoglucanase 1100-8000 nkat/g glucan,β-glucosidase 3000-15000 nkat/g glucan, endoxylanase 9000-30000 nkat/gglucan, β-xylosidase 75-1400 nkat/g glucan nkat/g glucan.

FIG. 13 shows cellulose conversion as a function of time for both of thewhole slurry hydrolysis samples. The cellulose conversion is determinedas described in example 9. As shown, the cellulose conversion kineticsare equivalent for the CTEC and TRIO chambers. Further, notwithstandinga lower enzyme dose, the conversion levels are equivalent with thoseachieved with C5 bypass and posthydrolysis, as described in example 9.

FIG. 14 shows corresponding xylan conversion as a function of time forboth of the whole slurry hydrolysis samples. As shown, the xylanconversion kinetics are approximately equivalent for the CTEC and TRIOchambers. Where xylan recovery from pretreatment was 77%, the xylanrecoveries shown in FIG. 14 correspond to very high final C5 monomerrecovery in the hydrolysate, for example, 80% conversion in FIG. 12corresponds to (0.80)*(0.77)=61.6% C5 monomer recovery. As shown, inwhole slurry hydrolysis, very high C5 monomer recoveries of at least55%, corresponding to 71% xylan conversion in FIG. 14, are achievedwithin 41 hours hydrolysis under these conditions.

The various parameters of pretreatment, hydrolysis and recovery for theexperiments described in examples 9 and 10 are shown in Table 4.

TABLE 4 Hydrolysis yields using very low severity single-stageautohydrolysis. Chamber 1 Chamber 2 Chamber 3 Chamber 4 Chamber 5Chamber 6 Biomass Wheat straw Wheat straw Wheat straw Wheat straw Wheatstraw Wheat straw Process w/C5 bypass w/C5 bypass w/C5 bypass w/C5bypass Whole slurry Whole slurry Dry matter after soaking 40 40 40 40 4040 [wt %] Residence time [min] 12 12 12 12 12 12 Temperature [° C.] 183183 183 183 183 183 Pretreatment severity 3.51 3.51 3.51 3.51 3.51 3.51[logRo] C5 recovery from 77 77 77 77 77 77 pretreatment [%] Xylan number13.5 13.5 13.5 13.5 13.5 13.5 Enzyme type CTec3 CTec3 Celluclast/ AcTRIO CTec3 Ac TRIO Novozym188 Enzyme dosage [FPU/g 14.3 14.3 14.3 14.310.7 10.7 glucan] % TS in fiber hydrolysis 22 22 22 22 12 12 Celluloseconversion after 77% 78% 49% 74% 71% 70% 96 h hydrolysis Hemicelluloseconversion 80% 82% 57% 76% 75% 71% after 96 h hydrolysis % TS in secondhydrolysis 18.5 18.5 18.5 18.5 Cellulose conversion after 73% 74% 48%76% 74% 74% 144 h Hemicellulose conversion 79% 79% 52% 79% 84% 80% after144 h Overall cellulose conversion 73% 74% 48% 76% 74% 74% Overall C5monomer yield 61% 61% 40% 61% 65% 62%

Example 11 Whole Slurry Hydrolysis of Pretreated Sugar Cane Bagasse

Sugar cane bagasse was pretreated as described in example 4 to severitylog Ro 3.43 (180° C. for a residence time of 12 minutes) to produce apretreated biomass having xylan number 12.0%, with approximately 6.8% byweight xylan remaining in undissolved solids, as estimated from FIG. 2.Xylan recovery from pretreatment was 83%. After leaving the reactor, theslurry was pressed to a fibre fraction of approximately 57% and a liquidfraction. The pretreated material, fibre fraction as well as liquidfraction, was collected and analysed. The dry matter and composition ofthe samples were determined as described in previous examples.

The experiments were conducted in the 6-chamber reactor described inexample 6. The pretreated bagasse was used as whole slurry mixture,where the fiber fraction was mixed with the liquid fraction beforeenzyme addition. The dry matter content was then adjusted with water to18 wt-% DM. The hydrolysis was carried out at 50° C. with a pH adjustedto between 4.7 and 5.3 with the use of 20 wt-% calcium hydroxidesolution (Ca(OH)₂). Accellerase Trio was used as enzyme in aconcentration of 0.16 mL/g glucan (9.5 FPU/g glucan). Each day a samplewas taken and analysed for sugar content. After 118 h the hydrolysis wasstopped. The experiments were performed in double determination.

Enzyme activities in nkat/g glucan, to be measured as described herein,that were used in the experiment were estimated (based on the valuesgiven in example 9) to be initially as follows:

Exogluc endogluc beta-gluc xylanase xylosidase Trio 478 1973 5591 8387186

It can be shown that the enzyme activities in these experiments measuredas described herein were initially within the range exoglucanase nkat/gglucan, endoglucanase nkat/g glucan, β-glucosidase nkat/g glucan,endoxylanase nkat/g glucan, β-xylosidase nkat/g glucan nkat/g glucan.

The various parameters of pretreatment, hydrolysis and recovery for theexperiments described in this examples 11 are shown in Table 5.

TABLE 5 Hydrolysis yields using very low severity single-stageautohydrolysis. Bagasse whole slurry Biomass Sugarcane bagasse Drymatter after soaking [wt %] 40.6%   Residence time [min] 12 minTemperature [° C.] 180 Pretreatment severity [logRo]    3.43 C5 recoveryfrom pretreatment [%] 83% Xylan number 12% Enzyme type Accellerase TRIOEnzyme dosage [g/kg glucan] 167 % DM in whole slurry hydrolysis 18%Cellulose conversion after 118 h hydrolysis 63% Hemicellulose conversionafter 118 h hydrolysis 75% Overall cellulose conversion 63% Overall C5monomer yield 62%

FIG. 15 shows total C5 and C6 monomer recovery in hydrolysate as afunction of hydrolysis time for the whole slurry hydrolysis of bagasse.As shown, total C5 monomer recovery of at least 55% is achieved within24 hours under these conditions.

Example 12 Improved Biomethane Potential of Thin Stillage Remainingafter C6 Ethanol Fermentation of Hydrolysate from Feedstock Pretreatedby Low Severity Autohydrolysis

Wheat straw was pretreated as described in example 4 to three differentseverities so as to produce pretreated biomass having xylan number 3.0%,9.1% and 13.2%, having respectively approximately 2.0%, 4.3% and 7.8% byweight xylan remaining in undissolved solids, as estimated from FIG. 2.

The pretreated material was subject to solid/liquid separation toproduce a fiber fraction, which was subsequently used in enzymatichydrolysis experiments, as well as a liquid fraction, which was keptseparate from the hydrolysis. The solid fractions were hydrolysed usingCTEC3 in the respective doses 0.052 g/g glucan for the 3.0% xylan numbersample, 0.056 g/g glucan for the 9.1% xylan number sample, and 0.075 g/gglucan for the 13.2% xylan number sample.

It can be shown that the enzyme activities in these experiments measuredas described herein were initially within the range exoglucanase nkat/gglucan, endoglucanase nkat/g glucan, β-glucosidase nkat/g glucan,endoxylanase nkat/g glucan, β-xylosidase nkat/g glucan nkat/g glucan.

Hydrolysis was conducted at 22% DM for 144 hours at 50° C. with pHadjusted to 5.0. After 144 hours, ordinary bakers' yeast (C6 fermenting)was added to the hydrolysate, the temperature lowered to 37° C. andfermentation/hydrolysis continued for another 60 hours. At the end ofthe hydrolysis/fermentation, ethanol concentrations were approximatelyequivalent between the different groups, between 61.77-63.68 g/kgethanol.

Biomethane production from the fermented hydrolysates and from thecorresponding separated liquid fraction was determined in duplicatebatch assays, using inoculum from Fredericia Spildevand, Fredericia,Denmark, at an inoculum/substratio ratio between 5-6.5 (on a volatilesolids basis).

Mass balances for each of the pretreatments were carefully determinedand used to estimate the methane potential of thin stillage remainingfrom C6 ethanol fermentation of each of hydrolysates from each of thethree different severity levels. Subtracting the known contribution ofethanol to observed biomethane potentials, estimate methane productionfrom 1 ton of wheat straw dry matter were determined. Results are shownin Table 6.

TABLE 6 Methane production from thin stillage and liquid fractionderived from 1 tonne of straw dry matter, depending on severity ofpretreatment. 13.2% (82% 13.2% (74% Xylan number 3.0% 9.1% conversion)conversion) C5 liquid [ton] 0.13 0.18 0.18 0.18 Thin stillage [ton]0.128 0.149 0.221 0.214 CH4 Nm3/ton straw 26.36 38.50 47.88 47.88 fromC5 liquid CH4 Nm3/ton straw 16.86 31.54 45.84 47.65 from thin stillageCH4 Nm3/ton straw 43 70 94 96 Total

The embodiments and examples are descriptive only and not intended tolimit the scope of the claims. Each of the references cited herein ishereby expressly incorporated by reference in entirety.

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The invention claimed is:
 1. A method of processing lignocellulosicbiomass comprising: Pretreating soft lignocellulosic biomass feedstockat pH within the range 3.5 to 9.0 in a single-stage pressurizedhydrothermal pretreatment reactor at a temperature of at least 140° C.for 1 to 60 minutes to log severity Ro 3.75 or lower to produce apretreated biomass slurry in which the undissolved solids comprise atleast 5.0% by weight xylan, and Hydrolysing the pretreated biomassslurry as a whole slurry with or without additional water content usingenzymatic hydrolysis for at least 24 hours catalysed by an enzymemixture comprising endoglucanase, exoglucanase, β-glucosidase,endoxylanase, and β-xylosidase activities at activity levels measured innkat/g glucan, wherein endoglucanase is at least 1100 nkat/g glucan,exoglucanase is at least 280 nkat/g glucan, β-glucosidase is at least3000 nkat/g glucan, endoxylanase isat least 1400 nkat/g glucan, andβ-xylosidase is at least 75 nkat/g glucan, to produce a hydrolysate inwhich the yield of C5 monomers is at least 55% of the original xyloseand arabinose content of the soft lignocellulosic biomass feedstockprior to pretreatment.
 2. The method of claim 1, wherein thehydrothermal pretreatment is conducted as an autohydrolysis, in whichacetic acid liberated by hemicellulose hydrolysis during pretreatmentfurther catalyzes hemicellulose hydrolysis.
 3. The method of claim 1,wherein the feedstock is subject to pressurized pretreatment at a drymatter content of at least 35% or at temperature between 160 and 200° C.4. The method of claim 1, wherein the whole slurry hydrolysate is usedas a biomethane substrate.
 5. The method of claim 1, wherein theenzymatic hydrolysis is conducted at dry matter content between 8 and19%.
 6. The method of claim 4, wherein the enzymatic hydrolysis isconducted at dry matter content of 20% or greater.
 7. The method ofclaim 1, wherein the endoglucanase activity is within the range1100-30000 nkat/g glucan, the exoglucanase activity is within the range280-20000 nkat/g glucan, the β-glucosidase activity is within the range3000-50000 nkat/g glucan, the endoxylanase activity is within the range1400-70000 nkat/g glucan and/or the β-xylosidase activity is within therange 75-20000 nkat/g glucan.
 8. The method of claim 7, wherein theβ-glucosidase activity is within the range 4000-50000 nkat/g glucan, theendoxylanase activity is within the range 4000-70000 nkat/g glucanand/or the β-xylosidase activity is within the range 250-20000 nkat/gglucan.
 9. The method of claim 1, wherein the pretreated biomass has afiber structure that is preserved during pretreatment.
 10. The method ofclaim 1, wherein the biomass feedstock is subject to particle sizereduction and/or other mechanical processing prior to hydrothermalpretreatment.
 11. The method of claim 1, wherein the pretreated biomassis subject to a solid/liquid separation step to produce a liquidfraction and a solid fraction having a dry matter content of at least40% by weight and the solid fraction is hydrolysed.
 12. The method ofclaim 4, wherein the separated liquid fraction is added back into thehydrolysis mixture, after enzymatic hydrolysis of the solid fraction hasreached a desired degree of glucan conversion.
 13. The method of claim1, wherein the feedstock is wheat straw, corn stover, sugar canebagasse, sweet sorghum bagasse, or empty fruit bunches.
 14. The methodof claim 1, wherein pressurized pretreatment is conducted at a pressureof 10 bar or lower.
 15. The method of claim 1, further comprisingremoving the feedstock from the pressurized hydrothermal pretreatmentreactor using a hydrocyclone system.
 16. The method of claim 1, whereinenzymatic hydrolysis is conducted for at least 96 hours.
 17. The methodof claim 1, further characterized in that the hydrolysate is a C5/C6hydrolysate and the C5/C6 hydrolysate is directly fermented to ethanol.18. The method of claim 17, wherein the ethanol fermentation uses one ormore modified yeast strains.